Vaccines

ABSTRACT

The present invention relates to particles, particularly virus-like particles (VLPs), comprising fusion polypeptides comprising selected repeat units derived from the repeating regions of Type I and Type II circumsporozoite proteins (CSP) of  Plasmodium vivax  (Pv), together with an amino acid sequence derived from the C-terminal PvCSP sequence. In some embodiments, the fusion polypeptide additionally comprises an amino acid sequence derived from the N-terminal PvCSP sequence and/or a surface antigen polypeptide derived from Hepatitis B virus (HBV-S). The invention also relates to nucleotide sequences coding for such fusion polypeptides, vectors and plasmids comprising such nucleotide sequences, and host cells comprising such vectors and plasmids. The invention additionally relates to compositions, particularly vaccine compositions, comprising the fusion polypeptides or VLPs for use as vaccines for the prevention of malaria.

The present invention relates to particles, particularly virus-like particles (VLPs), comprising fusion polypeptides comprising selected repeat units derived from the repeating regions of Type I and Type II circumsporozoite proteins (CSP) of Plasmodium vivax (Pv), together with an amino acid sequence derived from the C-terminal PvCSP sequence. In some embodiments, the fusion polypeptide additionally comprises an amino acid sequence derived from the N-terminal PvCSP sequence and/or a surface antigen polypeptide derived from Hepatitis B virus (HBV-S). The invention also relates to nucleotide sequences coding for such fusion polypeptides, vectors and plasmids comprising such nucleotide sequences, and host cells comprising such vectors and plasmids. The invention additionally relates to compositions, particularly vaccine compositions, comprising the fusion polypeptides or VLPs for use as vaccines for the prevention of malaria.

Plasmodium vivax malaria poses a major health risk to 2.85 billion people worldwide living in tropical, sub-tropical and temperate regions. P. vivax is the most widely distributed human malaria parasite in the world and considered to be the most difficult to control and eliminate from endemic regions. This is largely due to the parasite's ability to remain latent in the liver of infected people and to subsequently reactivate, possibly weeks to years after an initial infection.

Plasmodium sporozoites enter the blood stream through an infectious mosquito bite and quickly migrate and invade the liver. This ‘pre-erythrocytic’ stage is an attractive vaccine candidate. Currently, RTS,S/AS01 is the most advanced platform, aiming at preventing P. falciparum infection by stimulating immune responses against the major Plasmodium sporozoite surface antigen, i.e. the circumsporozoite protein (CSP). RTS,S/AS01 presents a hybrid polypeptide consisting of a portion of the P. falciparum CSP repeat and C-terminal regions fused to the amino terminal end of the hepatitis B virus surface (HepB-S) protein. RTS,S/AS01 has undergone extensive clinical testing and has been shown to induce significant protective immunity in Phase III trials.

The homologous CSP protein of P. vivax (PvCSP) is also actively being investigated as a component of a pre-erythrocytic vaccine against P. vivax. Some of these vaccines include the PvCSP VK210 (Type I) and VK247 (Type II) central repeats to try to induce protection against both types of parasites circulating worldwide.

WO2008/009652 relates to novel hybrid/fusion proteins comprising repeat units derived from the repeating regions of Type I and Type II CSPs of P. vivax, the N-terminal PvCSP sequence and a surface antigen S derived from Hepatitis B virus (HBV). The claims of this patent application refer to 1-15 Type I repeats; and 5 or less Type II repeats.

WO2009/080803 relates to immunogenic protein particles comprising a fusion protein comprising sequences derived from a CS protein of P. vivax, the N-terminal PvCSP sequence and the S antigen of HBV, together with an S antigen derived from HBV. This is referred to therein as CSV-S,S.

There remains a need, however, for improved antigens for use in inducing protective immunity against malaria.

A new fusion polypeptide comprising certain selected Type I and Type II repeat units from the CSPs of P. vivax and an amino acid sequence derived from the C-terminal PvCSP sequence has now been developed. In some embodiments, this fusion polypeptide additionally comprises an amino acid sequence derived from the N-terminal PvCSP sequence and/or a surface antigen polypeptide derived from Hepatitis B virus (HBV-S). Fusion polypeptides which comprise the surface antigen polypeptide derived from Hepatitis B virus (HBV-S) can be used to present the PvCSP antigen in a virus like particle (VLP); this is referred to herein as Rv21.

It is shown herein that vaccination with the fusion polypeptide antigen of the current invention induces protective immune responses against sporozoites expressing either one of the PvCSP VK210 or VK247 alleles. Moreover, it has been found that an immunization protocol using Rv21 VLPs induced complete, sterile protection against a stringent sporozoite challenge, surpassing the efficacy of viral-vectored vaccines expressing a similar PvCSP antigen. These studies highlight the potential and utility of Rv21 VLPs to support the development of an efficacious P. vivax malaria vaccine.

It is therefore an object of the invention to provide an immunogenic fusion polypeptide and VLPs comprising such polypeptides which are capable of inducing protective and/or neutralising antibodies against a sporozoite challenge.

In one embodiment, the invention provides a particle comprising a fusion polypeptide, wherein the fusion polypeptide comprises:

-   (i) at least 6 repeat units derived from the repeating region of a     Type I circumsporozoite protein (CSP) of Plasmodium vivax; -   (ii) at least 6 repeat units derived from the repeating region of a     Type II circumsporozoite protein (CSP) of Plasmodium vivax; -   (iii) an amino acid sequence of or derived from the C-terminal     fragment of CSP of Plasmodium vivax; and -   (iv) an amino acid sequence of or derived from the Hepatitis B virus     surface antigen.

The invention also provides a fusion polypeptide, wherein the fusion polypeptide comprises:

-   (i) at least 6 repeat units derived from the repeating region of a     Type I circumsporozoite protein (CSP) of Plasmodium vivax; -   (ii) at least 6 repeat units derived from the repeating region of a     Type II circumsporozoite protein (CSP) of Plasmodium vivax; and -   (iii) an amino acid sequence of or derived from the C-terminal     fragment of CSP of Plasmodium vivax.

In some embodiments, the fusion polypeptide additionally comprises an amino acid sequence of or derived from the N-terminal fragment of CSP of Plasmodium vivax and/or an amino acid sequence of or derived from the Hepatitis B virus surface antigen.

Preferably, the fusion polypeptide comprises:

-   (i) 8-12 repeat units derived from the repeating region of a Type I     circumsporozoite protein (CSP) of Plasmodium vivax; and/or -   (ii) 8-12 repeat units derived from the repeating region of a Type     II circumsporozoite protein (CSP) of Plasmodium vivax.

The fusion polypeptide comprises at least three parts, i.e. parts (i)-(iii); in some embodiments it has at least four parts (i.e. (i)-(iv)) or more. These parts are either joined contiguously or short linker amino acids may be used to join the parts together.

The parts may or may not be joined in the order (i)-(iii) or (i)-(iv) (N- to C-orientation). Preferably, they are joined in the order (i)-(iii) or (i)-(iv) (N- to C-orientation) with no linkers.

The circumsporozoite protein (CSP) of Plasmodium species is characterized by a central domain (repeat region) flanked by non-repetitive amino (N-terminus) and carboxy (C-terminus) fragments.

The central domain of P. vivax is composed of several blocks of a repeat unit. Generally each repeat unit consists of 9-amino acids.

At least two forms of the P. vivax CSP are known: these are designated VK210 or Type I; and VK247 or Type II. These two forms are exemplified by SEQ ID NOs: 1-2.

As used herein, the term “Type I repeat units” refers to repeat units derived from the repeating region of a Type I circumsporozoite protein (CSP) of Plasmodium vivax. Similarly, as used herein, the term “Type II repeat units” refers to repeat units derived from the repeating region of a Type II circumsporozoite protein (CSP) of Plasmodium vivax.

In some embodiments, the Type I repeat unit is derived from one or more of the following strains of P. vivax: KCSP95-50, KCSP96-21, KCSP97-75, North Korea, C-2, CH-3, CH-4, CH-5, NYUThai, India VII, PH-46, PH-79, SOL-83, SOL-101, Mauritania, Sal-1, Brazil or Belem.

In some embodiments, the Type II repeat unit is derived from one or more of the following strains of P. vivax: Papua New Guinea (PNG, PNG 4/B), Brazil: Paragaminos (BZL B7-4, BZL B19-2) Brazil (P. simium), Gabon or Bangladesh.

Examples of Type I repeat units include the following:

GDRAAGQPA (SEQ ID NO: 3)

GDRADGQPA (SEQ ID NO: 4)

GNGAGGQAA (SEQ ID NO: 5)

Examples of Type II repeat units include the following:

ANGAGNQPG (SEQ ID NO: 6)

ANGAGGQAA (SEQ ID NO: 7)

ANGAGDQPG (SEQ ID NO: 8)

ANGADDQPG (SEQ ID NO: 9)

EDGAGNQPG (SEQ ID NO: 10)

The invention also relates to derivatives of the repeat units disclosed herein wherein one or more of the repeat unit sequences independently comprise 1, 2, 3, 4 or 5 conservative amino acid modifications. The modifications may or may not be present in all of the repeat units. As used herein, the term “conservative sequence modifications” is intended to refer to amino acid modifications that do not significantly affect or alter ability of the fusion polypeptide to induce protective immunity against P. vivax CSP. Such conservative modifications include amino acid substitutions, additions and deletions. Conservative amino acid substitutions are ones in which the amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, one or more amino acids of the repeat units may be replaced with another amino acid from the same side chain family, and the modified amino acid sequence may be tested to evaluate its ability to induce protective immunity against P. vivax CSP.

In some embodiments, one or more of the repeat unit sequences independently comprise 1 or 2 conservative amino acid substitutions. In some embodiments, one or more of the repeat unit sequences independently comprise 1 or 2 amino acid additions or deletions.

Preferably, the Type II repeat units are present as one or more of the following repeat unit pairs:

(SEQ ID NO: 11) ANGAGNQPG-ANGAGGQAA (SEQ ID NO: 12) ANGAGDQPG-ANGAGDQPG (SEQ ID NO: 13) ANGADDQPG-ANGAGDQPG (SEQ ID NO: 14) EDGAGNQPG-ANGAGDQPG

Preferably, the at least 6 Type I repeat units comprise 5 blocks of the repeat unit GDRAAGQPA (SEQ ID NO: 3), more preferably 5 contiguous repeat units.

Preferably, the at least 6 Type I repeat units comprise 4 blocks of the repeat unit GDRADGQPA (SEQ ID NO: 4), more preferably 4 contiguous repeat units.

Preferably, the at least 6 Type I repeat units comprise 1 block of the repeat unit GNGAGGQAA (SEQ ID NO: 5).

Preferably, the at least 6 Type II repeat units comprise 2 blocks of the repeat unit pair ANGAGNQPG-ANGAGGQAA (SEQ ID NO: 11), more preferably 2 contiguous repeat units pairs.

Preferably, the at least 6 Type II repeat units comprise 1 block of the repeat unit pair ANGAGDQPG-ANGAGDQPG (SEQ ID NO: 12).

Preferably, the at least 6 Type II repeat units comprise 1 block of the repeat unit pair ANGADDQPG-ANGAGDQPG (SEQ ID NO: 13).

Preferably, the at least 6 Type II repeat units comprise 1 block of the repeat unit pair EDGAGNQPG-ANGAGDQPG (SEQ ID NO: 14).

Preferably, the at least 6 repeat units derived from the repeating region of a Type I circumsporozoite protein (CSP) of Plasmodium vivax comprise the following numbers of the following repeat units:

(SEQ ID NO: 3) (GDRAAGQPA)*5 (SEQ ID NO: 4) (GDRADGQPA)*4 (SEQ ID NO: 5) (GNGAGGQAA)*1 wherein the above repeat units are preferably joined contiguously in the above order in a N-C orientation.

As used herein, the term “at least 6 repeat units derived from the repeating region of a Type I circumsporozoite protein (CSP) of Plasmodium vivax” includes amino acid sequences having at least 80% sequence identity to SEQ ID NO: 16 (preferably at least 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity) and having the ability to induce protective immunity against P. vivax CSP.

As used herein, the term “at least 6 repeat units derived from the repeating region of a Type I circumsporozoite protein (CSP) of Plasmodium vivax” also includes fragments of SEQ ID NO: 16 which are at least 50% of the length of SEQ ID NO: 16 (preferably at least 60%, 70%, 80% or 90% of the length) and having the ability to induce protective immunity against P. vivax CSP.

Preferably, the at least 6 repeat units derived from the repeating region of a Type II circumsporozoite protein (CSP) of Plasmodium vivax comprise the following numbers of the following repeat units:

(SEQ ID NO: 11) (ANGAGNQPG/ANGAGGQAA)*2 (SEQ ID NO: 12) (ANGAGDQPG/ANGAGDQPG)*1 (SEQ ID NO: 13) (ANGADDQPG/ANGAGDQPG)*1 (SEQ ID NO: 14) (EDGAGNQPG/ANGAGDQPG)*1 wherein the above repeat units are preferably joined contiguously in the above order in a N-C orientation.

As used herein, the term “at least 6 repeat units derived from the repeating region of a Type II circumsporozoite protein (CSP) of Plasmodium vivax” includes amino acid sequences having at least 80% sequence identity to SEQ ID NO: 18 (preferably at least 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity) and having the ability to induce protective immunity against P. vivax CSP.

As used herein, the term “at least 6 repeat units derived from the repeating region of a Type II circumsporozoite protein (CSP) of Plasmodium vivax” also includes fragments of SEQ ID NO: 18 which are at least 50% of the length of SEQ ID NO: 18 (preferably at least 60%, 70%, 80% or 90% of the length) and having the ability to induce protective immunity against P. vivax CSP.

Most preferably, the invention provides a fusion polypeptide comprising an amino acid sequence comprising the following repeat units:

(SEQ ID NO: 3) (GDRAAGQPA)*5 (SEQ ID NO: 4) (GDRADGQPA)*4 (SEQ ID NO: 5) (GNGAGGQAA)*1 (SEQ ID NO: 11) (ANGAGNQPG-ANGAGGQAA)*2 (SEQ ID NO: 12) (ANGAGDQPG-ANGAGDQPG)*1 (SEQ ID NO: 13) (ANGADDQPG-ANGAGDQPG)*1 (SEQ ID NO: 14) (EDGAGNQPG-ANGAGDQPG)*1 wherein the above repeat units are joined contiguously in the above order in a N-C orientation.

As used herein, the term “*5”, etc., means multiplied by 5, etc., i.e. 5 contiguous repeated blocks of that sequence. The same applies to “*4”, “*2” and “*1”, mutatis mutandis.

In some embodiments, the fusion polypeptide as defined herein additionally comprises at least one repeat unit derived from the repeating region of a circumsporozoite protein (CSP) of Plasmodium vivax-like malaria parasite.

Plasmodium vivax-like malaria parasites are described, for example, in Shoukat et al. [60].

Preferably, the amino acid sequence of the repeat unit is APGANQ(E/G)GGAA (SEQ ID NO: 19).

Most preferably, the repeat unit is APGANQEGGAA (SEQ ID NO: 20) or APGANQGGGAA (SEQ ID NO: 21).

Preferably, the fusion polypeptide comprises 1-10, most preferably 4-8, even more preferably 6 of these repeat units.

Most preferably, the fusion polypeptide comprises:

(SEQ ID NO: 20) (APGANQEGGAA)*3 (SEQ ID NO: 21) (APGANQGGGAA)*3.

As used herein, the term “at least one repeat unit derived from the repeating region of a circumsporozoite protein (CSP) of Plasmodium vivax-like malaria parasite” includes amino acid sequences having at least 80% sequence identity to one or more of SEQ ID 23 or 25-27 (preferably at least 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity) and having the ability to induce protective immunity against P. vivax-like CSP.

As used herein, the term “at least one repeat unit derived from the repeating region of a circumsporozoite protein (CSP) of Plasmodium vivax-like malaria parasite” also includes fragments of SEQ ID NOs: 29-32 which are at least 50% of the length of SEQ ID NOs: 29-32 (preferably at least 60%, 70%, 80% or 90% of the length) and having the ability to induce protective immunity against P. vivax-like CSP.

These Plasmodium vivax-like repeat units are preferably placed adjacent to the other repeat units, i.e. downstream of the N-terminal CSP sequence (if present) and upstream of the C-terminal CSP sequence. Most preferably, these Plasmodium vivax-like repeat sequences are positioned immediately downstream of the P. vivax Type II repeat units, and immediately upstream of the C-terminal CSP sequence.

The fusion polypeptide also comprises (iii) an amino acid sequence of or derived from the C-terminal fragment of CSP of Plasmodium vivax.

The complete C-terminal fragment from CSP of Plasmodium vivax is given in SEQ ID NO: 29. Preferably, the C-terminal fragment consists of or comprises SEQ ID NO: 29.

More preferably, the amino acid sequence derived from the C-terminal fragment of CSP of Plasmodium vivax comprises or consists of SEQ ID NO: 30,

or

EWTPCSVTCG (SEQ ID NO: 31)

or

CSVTCG (SEQ ID NO: 32).

The fusion polypeptide may also comprise an insertion of the sequence GAGGQAAGGNA (SEQ ID NO: 33) in the insertion region (IR) of the CSP. The IR lies between the central repeat region and the C-terminal sequence.

The amino acid sequence derived from the C-terminal fragment of CSP of Plasmodium vivax is preferably positioned in the fusion polypeptide downstream of the blocks of Type II repeat units (and downstream of the P. vivax-like repeat sequences, when present), and before the HBV S antigen sequence (when present).

As used herein, the term “amino acid sequence derived from the C-terminal fragment of CSP” includes amino acid sequences having at least 80% sequence identity to one or more of SEQ ID NOs: 29-32 (preferably at least 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity) and comprising one or more T-cell epitopes which promote the immunogenicity of the Type I and/or Type II repeat units.

As used herein, the term “amino acid sequence derived from the C-terminal fragment of CSP” also includes fragments of SEQ ID NOs: 29-32 which are at least 50% of the length of SEQ ID NOs: 29-32 (preferably at least 60%, 70%, 80% or 90% of the length) and comprising one or more T-cell epitopes which promote the immunogenicity of the Type I and/or Type II repeat units.

In some embodiments of the invention, the fusion polypeptide additionally comprises an amino acid sequence of or derived from the N-terminal fragment of CSP of Plasmodium vivax or Plasmodium falciparum, preferably from P. vivax.

The complete N-terminal fragment from CSP of Plasmodium vivax is given in SEQ ID NO: 35. Preferably, the N-terminal fragment sequence comprises SEQ ID NO: 35.

More preferably, the N-terminal fragment comprises KLKQP (SEQ ID NO: 36).

The N-terminal fragment of CSP of Plasmodium vivax or Plasmodium falciparum is preferably positioned, when present, at the N-terminus of the fusion polypeptide (i.e. upstream of the blocks of repeats).

In some embodiments of the invention, the particle and fusion polypeptide does not comprise an amino acid sequence derived from the N-terminal fragment of CSP of Plasmodium vivax or Plasmodium falciparum.

In some particular embodiments of the invention, the particle and fusion polypeptide does not comprise an amino acid sequence of SEQ ID NO: 35 or a variant thereof having at least 90% sequence identity thereto.

As used herein, the term “amino acid sequence derived from the N-terminal fragment of CSP” includes amino acid sequences having at least 80% sequence identity to SEQ ID NO: 35 (preferably at least 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity) and comprising one or more T-cell epitopes which promote the immunogenicity of the Type I and/or Type II repeat units.

As used herein, the term “amino acid sequence derived from the N-terminal fragment of CSP” also includes fragments of SEQ ID NO: 35 which are at least 50% of the length of SEQ ID NO: 35 (preferably at least 60%, 70%, 80% or 90% of the length) and comprising one or more T-cell epitopes which promote the immunogenicity of the Type I and/or Type II repeat units.

The particle of the invention comprises (iv) an amino acid sequence of or derived from Hepatitis B virus surface antigen.

The HBV surface antigen (i.e. HBV SAg) increases the immunogenicity of the CSP portion of the fusion polypeptide.

In some embodiments, the HBV surface antigen is derived from an adw serotype.

The complete amino acid sequence of the HBV surface antigen derived from an adw serotype is given in SEQ ID NO: 38.

The invention extends to fusion polypeptides comprising an amino acid sequence derived from Hepatitis B virus surface antigen S as given in SEQ ID NO: 38.

Preferably, the amino acid sequence derived from the Hepatitis B virus surface antigen comprises or consists of the amino acid sequence given in SEQ ID NO: 38.

As used herein, the term “amino acid sequence derived from the Hepatitis B virus surface antigen” includes amino acid sequences having at least 80% sequence identity to SEQ ID NO: 38 (preferably at least 85%, 90%, 95%, 96%, 97%, 98% or 99%) and having the ability to increase the immunogenicity of parts (i)-(iii) of the fusion polypeptide of the invention.

As used herein, the term “amino acid sequence derived from the Hepatitis B virus surface antigen” also includes fragments of SEQ ID NO: 38 which are at least 80% of the length of SEQ ID NO: 38 (preferably at least 90%, 95%, 96%, 97%, 98% or 99%) and having the ability to increase the immunogenicity of parts (i)-(iii) of the fusion polypeptide of the invention.

The amino acid sequence derived from the Hepatitis B virus surface antigen must also be capable of forming virus-like particles (VLPs).

In one preferred embodiment, the invention provides a fusion polypeptide consisting of or comprising the amino acid sequence as given in SEQ ID NO: 39 or an amino acid sequence having at least 80% sequence identity thereto (preferably at least 90%, 95%, 96%, 97%, 98% or 99%) and having the ability to induce protective immunity against P. vivax CSP in a human subject.

In some embodiments, one more of the repeat units and/or the N- (when present) or C-terminal and/or HBV S antigen (when present) are independently separated from one another by one more linker amino acids, e.g. 1, 2, 3, 4 or 5 amino acids.

When present, the linker amino acids should not significantly affect (i.e. significantly reduce) the ability of the fusion polypeptide or VLP to elicit protective immunity in a subject (e.g. a human subject). Preferably, no linker amino acids are used.

In some embodiments, the fusion polypeptide additionally comprises a peptide sequence which facilitates the purification of the fusion polypeptide. This peptide sequence is preferably positioned at the C-terminus of the fusion polypeptide. The peptide sequence is preferably a C-tag sequence, more preferably comprising the sequence EPEA (SEQ ID NO: 43).

In some embodiments, the fusion polypeptide additionally comprises one or more further antigens derived from P. falciparum and/or P. vivax. The further antigen(s) may, for example, be selected from the group consisting of DBP, PvTRAP, PvMSP2, PvMSP4, PvMSP5, PvMSP6, PvMSP7, PvMSP8, PvMSP9, PvAMA1 and RBP.

Percentage amino acid sequence identities and nucleotide sequence identities may be obtained using the BLAST methods of alignment (Altschul et al. (1997), “Gapped BLAST and PSI-BLAST: a new generation of protein database search programs”, Nucleic Acids Res. 25:3389-3402; and http://www.ncbi.nlm.nih.gov/BLAST). Preferably the standard or default alignment parameters are used.

Standard protein-protein BLAST (blastp) may be used for finding similar sequences in protein databases. Like other BLAST programs, blastp is designed to find local regions of similarity. When sequence similarity spans the whole sequence, blastp will also report a global alignment, which is the preferred result for protein identification purposes. Preferably the standard or default alignment parameters are used. In some instances, the “low complexity filter” may be taken off.

BLAST protein searches may also be performed with the BLASTX program, score=50, wordlength=3. To obtain gapped alignments for comparison purposes, Gapped BLAST (in BLAST 2.0) can be utilized as described in Altschul et al. (1997) Nucleic Acids Res. 25: 3389. Alternatively, PSI-BLAST (in BLAST 2.0) can be used to perform an iterated search that detects distant relationships between molecules. (See Altschul et al. (1997) supra). When utilizing BLAST, Gapped BLAST, PSI-BLAST, the default parameters of the respective programs may be used.

With regard to nucleotide sequence comparisons, MEGABLAST, discontiguous-megablast, and blastn may be used to accomplish this goal. Preferably the standard or default alignment parameters are used. MEGABLAST is specifically designed to efficiently find long alignments between very similar sequences. Discontiguous MEGABLAST may be used to find nucleotide sequences which are similar, but not identical, to the nucleic acids of the invention.

The BLAST nucleotide algorithm finds similar sequences by breaking the query into short subsequences called words. The program identifies the exact matches to the query words first (word hits). The BLAST program then extends these word hits in multiple steps to generate the final gapped alignments. In some embodiments, the BLAST nucleotide searches can be performed with the BLASTN program, score=100, wordlength=12.

One of the important parameters governing the sensitivity of BLAST searches is the word size. The most important reason that blastn is more sensitive than MEGABLAST is that it uses a shorter default word size (11). Because of this, blastn is better than MEGABLAST at finding alignments to related nucleotide sequences from other organisms. The word size is adjustable in blastn and can be reduced from the default value to a minimum of 7 to increase search sensitivity.

A more sensitive search can be achieved by using the newly-introduced discontiguous megablast page (www.ncbi.nlm.nih.gov/Web/Newsltr/FallWinter02/blastlab.html). This page uses an algorithm which is similar to that reported by Ma et al. (Bioinformatics. 2002 March; 18(3): 440-5). Rather than requiring exact word matches as seeds for alignment extension, discontiguous megablast uses non-contiguous word within a longer window of template. In coding mode, the third base wobbling is taken into consideration by focusing on finding matches at the first and second codon positions while ignoring the mismatches in the third position. Searching in discontiguous MEGABLAST using the same word size is more sensitive and efficient than standard blastn using the same word size. Parameters unique for discontiguous megablast are: word size: 11 or 12; template: 16, 18, or 21; template type: coding (0), non-coding (1), or both (2).

In a further embodiment, the invention provides a nucleic acid molecule which codes for a fusion polypeptide of the invention. The nucleic acid molecule may be DNA or RNA, preferably DNA.

The invention also provides a vector or plasmid comprising a nucleic acid molecule of the invention. Preferably, the vector is an expression vector. The vector and/or plasmid may comprise one or more regulatory sequences which are operably linked to the sequence which encodes the fusion polypeptide, e.g. one or more enhancer, promoter and/or transcriptional terminator sequences.

Preferably, the vector is a non-replicating vector, e.g. a non-replicating viral vector.

In some preferred embodiments, the vector is an adenoviral vector or a Modified Vaccinia Ankara (MVA) viral vector.

Preferably, the adenovirus vector is selected from the group consisting of a human adenovirus vector, a simian adenovirus vector, a group B adenovirus vector, a group C adenovirus vector, a group E adenovirus vector, an adenovirus 6 vector, a PanAd3 vector, an adenovirus C3 vector, a ChAdY25 vector, an AdC68 vector and an Ad5 vector.

The invention also provides a host cell comprising a vector or plasmid of the invention. Preferably, the host cell is a eukaryotic host cell. Examples of eukaryotic host cells include yeast and mammalian cells. In some preferred embodiments, the host cell is a yeast cell (e.g. Saccharomyces cerevisiae).

In other preferred embodiments, the yeast is Pichia pastoris. Pichia pastoris is frequently used as an expression system for the production of proteins. A number of properties make P. pastoris suited for this task: P. pastoris has a high growth rate and is able to grow on a simple, inexpensive medium; and P. pastoris can grow in either shake flasks or a fermenter, which makes it suitable for both small and large scale production.

In other embodiments, the host cell is a methylotrophic yeast, e.g. Hansenula, preferably Hansenula polymorpha.

The nucleic acid molecule of the invention may be codon-optimised for expression in the host cell, e.g. yeast.

Once expressed in an appropriate system, fusion polypeptides of the invention which comprise a HBV surface antigen are able to assemble spontaneously into lipoprotein structures/particles composed of numerous monomers of said fusion polypeptides.

These particles may be referred to as Virus Like Particles (VLPs). Virus-like particles resemble viruses, but are non-infectious because they do not contain any viral genetic material. The particles may also be described as multimeric lipoprotein particles.

Thus in a further aspect, the invention provides a composition comprising a particle, preferably a VLP, which comprises one or more fusion polypeptides of the invention. The particle is preferably immunogenic.

The invention also provides a composition comprising a multimeric lipoprotein particle which comprises one or more fusion polypeptides of the invention.

In some embodiments of the invention, the particle additionally comprises an amino acid sequence of or derived from Hepatitis B virus surface antigen (in addition to that which is present in the fusion polypeptide). In such embodiments, the ratio of amino acid sequence derived from Hepatitis B virus surface antigen to fusion polypeptide may be from 0.1 to 1.0.

In other embodiments, the particle does not additionally comprise an amino acid sequence derived from Hepatitis B virus surface antigen (other than that which is present in the fusion polypeptide).

In some preferred embodiments, the particle comprises a CSP fragment:HBV S antigen ratio of approximately 1:1.

The invention also provides a VLP (e.g. a VLP comprising HBV surface antigen polypeptides) wherein one or more fusion polypeptides of the invention are covalently attached to the VLP. For example, the fusion polypeptides of the invention may be covalently attached to the VLP by using chemical cross-linkers, reactive unnatural amino acids or SpyTag/SpyCatcher reactions.

In a further embodiment, the invention provides a composition comprising a particle or fusion polypeptide of the invention, optionally together with one or more pharmaceutically-acceptable carriers, excipients or diluents.

The invention particularly provides a composition comprising a particle or fusion polypeptide of the invention, together with a pharmaceutically-acceptable adjuvant.

The invention also provides a vaccine composition comprising a particle or fusion polypeptide of the invention or a vector or plasmid of the invention, preferably a vaccine composition suitable for parenteral administration.

Examples of suitable adjuvants include those which are selected from the group consisting of:

-   -   metal salts such as aluminium hydroxide or aluminium phosphate,     -   oil in water emulsions,     -   toll like receptors agonist, (such as toll like receptor 2         agonist, toll like receptor 3 agonist, toll like receptor 4         agonist, toll like receptor 7 agonist, toll like receptor 8         agonist and toll like receptor 9 agonist),     -   saponins, for example Quil A and its derivatives such as QS7         and/or QS21,     -   CpG containing oligonucleotides,     -   3D-MPL,     -   (2-deoxy-6-o-[2-deoxy-2-[(R)-3-dodecanoyloxytetra-decanoylamino]-4-o-phosphono-[β-D-glucopyranosy]]-2-[(R)-3-hydroxytetradecanoylamino]-α-D-glucopyranosyldihydrogenphosphate),     -   DP         (3S,9R)-3-[(R)-dodecanoyloxytetradecanoylamino]-4-oxo-5-aza-9-(R)-[(R)-3-hydroxytetradecanoylamino]decan-1,10-diol,         1,10-bis(dihydrogenophosphate), and     -   MP-Ac DP         (3S-,9R)-3-[(R)-dodecanoyloxytetradecanoylamino]-4-oxo-5-aza-9-[(R)-3-hydroxytetradecanoylamino]decan-I,         10-diol, 1-dihydrogenophosphate 10-(6-aminohexanoate),         or combinations thereof.

Preferably, the adjuvant is selected from the group comprising:

-   -   a saponin associated with a metallic salt, such as aluminium         hydroxide or aluminium phosphate     -   3D-MPL, QS21 and a CpG oligonucleotide, for example as an oil in         water formulation,     -   saponin in the form of a liposome, for example further comprise         a sterol such as QS21 and sterol, and     -   ISCOM.

In some particularly preferred embodiments, the adjuvant comprises a saponin. Saponins are steroid or triterpenoid glycosides, which occur in many plant species. Saponin-based adjuvants act in part by stimulating the entry of antigen-presenting cells into the injection site and enhancing antigen presentation in the local lymph nodes.

Preferably, the adjuvant comprises saponin, cholesterol and a phospholipid, e.g. ISCOM Matrix-M™ (Isconova, Novavax).

In Matrix-M, purified saponin fractions are mixed with synthetic cholesterol and a phospholipid to form stable particles than can be readily formulated with a variety of vaccine antigens. Matrix-M™ induces both a cell-mediated and an antibody mediated immune response.

In some other preferred embodiments, the adjuvant comprises a squalene-oil-in-water nano-emulsion emulsion, e.g. AddaVax™ (InvivoGen).

Squalene is an oil which is more readily metabolized than the paraffin oil used in Freund's adjuvants. Squalene oil-in-water emulsions are known to elicit both cellular (Th1) and humoral (Th2) immune responses. This class of adjuvants is believed to act through recruitment and activation of APC and stimulation of cytokines and chemokines production by macrophages and granulocytes.

In some other preferred embodiments, the adjuvant comprises two immunostimulants: 3-O-desacyl-4′-monophosphoryl lipid A (MPL) or a variant thereof, and a saponin, preferably QS-21.

In some particularly-preferred embodiments, the adjuvant is a liposome-based adjuvant.

In some particularly preferred embodiments, the adjuvant is AS01. AS01 is a liposome-based vaccine adjuvant system containing two immunostimulants: 3-O-desacyl-4′-monophosphoryl lipid A (MPL) and the saponin QS-21.

The composition may further comprise one or more additional antigens derived from P. falciparum and/or P. vivax in admixture.

The composition may further comprise a surfactant. Examples of suitable surfactants include Tween (such as Tween 20), briji and polyethylene glycol.

Vaccine preparation is generally described in New Trends and Developments in Vaccines, edited by Voller et al., University Park Press, Baltimore, Md., U.S.A., 1978. Encapsulation within liposomes is described, for example, by Fullerton, U.S. Pat. No. 4,235,877.

The amount of the particle or fusion polypeptide of the present invention present in each vaccine dose is selected as an amount which induces an immunoprotective response without significant, adverse side effects in typical vaccines. Such amount will vary depending upon which specific immunogen is employed and whether or not the vaccine is adjuvanted. Generally, it is expected that each does will comprise 1-1000 μg of protein, for example 1-200 μg such as 10-100 μg more particularly 10-40 μg. An optimal amount for a particular vaccine can be ascertained by standard studies involving observation of antibody titres and other responses in subjects. Following an initial vaccination, subjects will preferably receive a boost in about 4 weeks, followed by repeated boosts every six months for as long as a risk of infection exists. The immune response to the protein of this invention is enhanced by the use of adjuvant and or an immunostimulant.

The amount of saponin for use in the adjuvants of the present invention may be in the region of 1-1000 μg per dose, generally 1-500 μg per dose, more such as 1-250 μg per dose, and more specifically between 1 to 100 μg per dose (e.g. 10, 20, 30, 40, 50, 60, 70, 80 or 90 μg per dose).

One promising strategy for malaria control consists of targeting antigens expressed on the surface of the sexual stages of the malaria parasites, such as gametocytes, gametes, zygotes or ookinetes. Antibodies against such antigens can inhibit the development of the parasite within the mosquitoes, preventing transmission from the mosquitoes to humans.

Pvs25 and Pvs28 are proteins expressed on the surface of the Plasmodium vivax zygotes and ookinetes. Both proteins are essential for the survival of ookinetes in the mosquito midgut, and for the penetration of the midgut epithelium, supporting transformation into oocysts. Pvs25 has received the most attention and has been tested in Phase I clinical trials. Immunisation with this protein has raised antibodies with transmission-blocking activity. Similarly, antibodies raised in mice against Pvs28 have been shown to block the development of oocysts in mosquitoes, blocking transmission of the Sal I strain of P. vivax. Transmission-blocking activity was achieved against various natural isolates from Thailand, indicating that variability of the sequences is not an issue for this approach.

In yet a further embodiment, therefore, the invention provides a fusion polypeptide as defined herein which additionally comprises a transmission-blocking component.

Preferably, the transmission-blocking component is a P. vivax Pvs25 and/or Pvs28 polypeptide, or a variant or derivative thereof.

Preferably, the Pvs25 amino acid sequence is as given in SEQ ID NO: 45, or a variant or derivative thereof.

Preferably, the Pvs28 amino acid sequence is as given in SEQ ID NO: 46, or a variant or derivative thereof.

In some embodiments, the sequences of PVs25 and 28 are fused, e.g. as in SEQ ID NO: 47, or a variant or derivative thereof.

Preferably, the transmission blocking component is positioned at the N- and/or C-terminal of the Hepatitis B virus surface antigen.

In some embodiments, the fusion polypeptide comprises:

-   (i) a transmission-blocking component, preferably a P. vivax Pvs25     and/or Pvs28 polypeptide, or a variant or derivative thereof; -   (ii) an amino acid sequence of or derived from the Hepatitis B virus     surface antigen; -   (iii) at least 6 repeat units derived from the repeating region of a     Type I circumsporozoite protein (CSP) of Plasmodium vivax; -   (iv) at least 6 repeat units derived from the repeating region of a     Type II circumsporozoite protein (CSP) of Plasmodium vivax; -   (v) at least one repeat unit derived from the repeating region of a     circumsporozoite protein (CSP) of Plasmodium vivax-like malaria     parasite; -   (vi) an amino acid sequence of or derived from the C-terminal     fragment of CSP of Plasmodium vivax; and optionally -   (vii) a C-tag sequence,     preferably in the above N- to C-order.

In yet other embodiments, the fusion polypeptide comprises:

-   (i) at least 6 repeat units derived from the repeating region of a     Type I circumsporozoite protein (CSP) of Plasmodium vivax; -   (ii) at least 6 repeat units derived from the repeating region of a     Type II circumsporozoite protein (CSP) of Plasmodium vivax; -   (iii) at least one repeat unit derived from the repeating region of     a circumsporozoite protein (CSP) of Plasmodium vivax-like malaria     parasite; -   (iv) an amino acid sequence of or derived from the C-terminal     fragment of CSP of Plasmodium vivax; -   (v) an amino acid sequence of or derived from the Hepatitis B virus     surface antigen; -   (vi) a transmission-blocking component, preferably a P. vivax Pvs25     and/or Pvs28 polypeptide, or a variant or derivative thereof; and     optionally -   (vii) a C-tag sequence,     in the above N- to C-order.

In yet further embodiments, the invention provides a particle of the invention, a fusion polypeptide of the invention, a vector or plasmid of the invention or a composition of the invention for use in therapy or for use as a medicament.

In further embodiments, the invention provides a particle of the invention, a fusion polypeptide of the invention, a vector or plasmid of the invention, or a composition of the invention for use in the treatment/prevention of malaria, preferably wherein the malaria is caused by Plasmodium vivax.

In further embodiments, the invention provides the use of a particle of the invention, a fusion polypeptide of the invention, a vector or plasmid of the invention, or a composition of the invention in the manufacture of a medicament for use in the treatment or prevention of malaria, preferably wherein the malaria is caused by Plasmodium vivax.

The invention also provides a method of treating a patient susceptible to Plasmodium infection comprising administering an effective amount of a particle of the invention, a fusion polypeptide of the invention, a vector or plasmid of the invention, or a composition of the invention, particularly as a vaccine, thereby to treat or prevent malaria.

The particle, fusion polypeptide, vector or plasmid, or composition of the invention may also be used in similar uses and methods to produce neutralising antibodies in vivo against antigens on Plasmodium parasites, preferably on Plasmodium vivax.

The efficacy of the uses and methods to treat/prevent malaria may be tested (e.g. by ELISA) by establishing the presence or absence of neutralising antibodies against CSP in the patient's blood.

The invention also extends to prime-boost regimes. For example, priming and/or boosting may be effected with a P. vivax polypeptide (or a nucleic acid encoding such a polypeptide), a fusion protein of the invention or a particle of the invention.

The P. vivax polypeptide may, for example, be a TRAP polypeptide (also known as Sporozoite Surface Protein 2 or SSP2) or a variant, fragment or derivative thereof. In all embodiments disclosed herein, the P. vivax polypeptide (e.g. TRAP polypeptide) may be replaced by a nucleic acid (e.g. viral vector) encoding such a polypeptide.

The wild-type amino acid sequence of the P. vivax polypeptide TRAP polypeptide is given in SEQ ID NO: 40.

The amino acid sequence of a variant of the P. vivax TRAP polypeptide is given in SEQ ID NO: 41. In this variant, the first 22 amino acids have been replaced with the tPA amino acid sequence in order to facilitate secretion. Additionally, the C-terminal end has been truncated to facilitate secretion.

Preferably, the P. vivax polypeptide is an amino acid sequence derived from the polypeptide given in SEQ ID NO: 40 or 41.

The invention particularly provides a pharmaceutical preparation comprising a particle or fusion polypeptide of the invention together with a P. vivax polypeptide, preferably a TRAP polypeptide or variant, derivative or fragment thereof, as a combined preparation in a form suitable for simultaneous, separate or sequential use.

As used herein, the term “an amino acid sequence derived from the polypeptide given in SEQ ID NO: 40 or 41” includes amino acid sequences having at least 80% sequence identity to SEQ ID NO: 40 or 41 (preferably at least 85%, 90%, 95%, 96%, 97%, 98% or 99%) and having the ability to induce an immunogenic response against P. vivax.

As used herein, the term “amino acid sequence derived from the polypeptide given in SEQ ID NO: 40 or 41” also includes fragments of SEQ ID NOs: 40 or 41 which are at least 80% of the length of SEQ ID NO: 40 or 41 (preferably at least 90%, 95%, 96%, 97%, 98% or 99%) having the ability to induce an immunogenic response against P. vivax.

In some embodiments, the polypeptide, a fusion protein of the invention or a particle of the invention may be replaced by a nucleic acid molecule (preferably DNA) coding for the polypeptide, fusion protein or particle. The nucleic acid molecule may, for example, be a viral vector (e.g. adenoviral vector or MVA).

Other prime boost regimes are described below. For example:

-   -   priming may be with an antigen (such as an adjuvanted antigen)         comprising at least one Type I repeat unit from the CS protein         of P. vivax and at least one Type II repeat unit from the CS         protein of P. vivax; and boosting may be with a viral vector         encoding the same/corresponding antigen,     -   priming may be with an antigen (such as an adjuvanted antigen)         comprising at least one repeat unit or epitope from P. vivax and         at least one repeat unit or epitope from the CS protein of P.         falciparum; and boosting may be with a viral protein encoding         the same/corresponding antigen,     -   priming may be with a viral vector encoding an antigen         comprising at least one Type I repeat unit from the CS protein         of P. vivax and at least one Type II repeat unit from the CS         protein of P. vivax or comprising at least one repeat unit or         epitope from P. vivax and at least one repeat unit or epitope         from the CS protein of P. falciparum; and boosting with the         same/corresponding adjuvanted antigen in the form of a protein         and/or immunogenic particle.

Antigen in these contexts includes fusion protein and/or immunogenic particles of the invention. In these prime boost regimes, antigen will usually be adjuvanted.

In yet further embodiments, the invention provides a process for the production of a particle or fusion polypeptide of the invention, which process comprises expressing a nucleic acid molecule coding for said particle or polypeptide in a suitable host and recovering the product.

Preferably, the host is a yeast. More preferably, the host cell is Pichia pastoris or Saccharomyces.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1: Cellular immune responses following immunization with viral-vectored vaccines (vv; ChAd63 or MVA) expressing different versions of PvCSP. (a) Schematic representation of the CSP sequences expressed by ChAd63 and MVA vectors. NC: the amino (N)- and carboxy-terminal (C) sequences without central repeat regions (CR); a fragment containing the repeat sequences of the VK210 allele (N210CR) or the VK247 allele (N247CR) or a chimeric fragment with both repeat sequences (N210/247CR), all contain an insertion region (IR); (b) Groups of mice of four strains (C571316, C3H/HE, BALB/c, CD1, n=6) were immunized with different vv using an Ad prime- and MVA boost regimen at an interval of 8 weeks and cellular responses were assessed by ex vivo IFN-γ ELISpot on week two after the MVA boost. (c) Antigen-specific T-cell responses as measured by ex-vivo IFN-γ ELISPOT assay. Splenocytes were stimulated with four sub-pools of peptides overlapping the N-, 210-, 247-, and C-terminal region of the PvCSP protein.

FIG. 2. Antibody responses following immunization with viral-vectored vaccines (vv; ChAd63 or MVA) expressing different versions of PvCSP. Outbred CD-1 mice were immunized with vv expressing various versions of PvCSP (N210/N247C, N210C, N247C, NC; see FIG. 1) using an interval of 8 weeks. Control groups were mock-vaccinated with vv expressing P. berghei CSP and naive mice were injected with PBS. Serum was collected every two weeks and antibody titers were quantified using a standardized ELISA against the PvCSP VK210 and VK247 antigens.

FIG. 3. Immunofluorescence Assay (IFA) to assess CSP antibody reactivity of sera from immunized mouse on the surface of wild type P. vivax parasites isolated from Chiapas, Mexico. (A) Serum obtained from C57Bl/6 mice was incubated with P. vivax VK210 (top panel) or VK247 (bottom panel) sporozoites collected from the endemic state of Chiapas, Mexico. Each mouse group was immunized with ChAd63-MVA vv expressing homologous PvCSP transgenes (NC, N210C, N247C, N210/247C) and sera was obtained on week two after the final immunization. PBS and monoclonal antibodies with specificity to VK210 (Mab5D3) or VK247 (Mab2E10) were used as negative or positive controls, respectively. The experiment was performed using 6 mice/group and three batches of sporozoites for each sample.

FIGS. 4A-D: Cloning strategy for the development of two P. berghei parasite lines expressing P. vivax CSP VK210 or VK247.

FIG. 4A. Schematic representation describing the generation of PbANKA-PvCSP(r)_(PbCSP)(2196cl1 and 2199cl1) chimeric lines; where the P. berghei csp (Pbcsp) gene coding sequence (CDS) is replaced GIMO deletion-construct (construct 1; pL1929) is used to replace the Pbcsp CDS with the positive/negative selectable maker (SM; hdhfr::yfcu) cassette, resulting in the generation of the Pbcsp GIMO line (PbANKA-PbCSP GIMO; 2151cl1) after positive selection with pyrimethamine. Construct 1 targets the Pbcsp gene by double cross-over homologous recombination. Step 2: The GIMO insertion-construct (construct 2; pL1942 or pL1943) is used to replace the SM in the GIMO line with Pvcsp VK-210 or VK-247 CDS, respectively, after negative selection using 5-fluorocytosine (5-FC), resulting in the two chimeric lines PbANKA-PvCSP VK210(r)_(PbCSP) and PbANKA-PvCSP VK247(r)_(PbCSP) (2196cl1 and 2199cl1), respectively. Construct 2 integrates by double cross-over homologous recombination using the same targeting regions (TRs) employed in construct 1, resulting in the introduction of Pvcsp gene under the control of the Pbcsp gene promoter and transcriptional terminator sequences and removal of the SM.

FIG. 4B. Schematic representation of the reporter PbANKA parasite line PbGFP-LUC_(eef1α) (676m1cl1) which used to generate the replacement gene [RG] chimeric parasites. It expresses a fusion protein of GFP and firefly luciferase (LUC-IAV) under the constitutive Pbeef1a promoter and is selectable marker (SM) free. The reporter-cassette is integrated into the neutral 230p locus in chromosome-3.

FIG. 4C. Genotype analysis of Replacement Gene [RG] chimeric parasites and their intermediate GIMO mother-line knock out chimeric parasites using Southern analysis of chromosomes (chrs) separated by pulsed-field gel electrophoresis (PFGE) Left panel: PbANKA-ΔCSP GIMO: 2151 cl1. The correct integration of the SM in the right locus in Chr-4 and replacing the endogenous PbCSP gene (PBANKA_040320) was confirmed by using the 3′UTR Pbdhfr/ts probe which hybridized Chr-3 because of the presence of the GFP-Luc cassette with 3′UTR Pbdhfr/ts, hybridized Chr-4 which confirms the corrected integration of the SM cassette into the right locus and replacing PbCSP gene with the SM and also hybridizes to the endogenous Pbdhfr/ts on Chr. 7. The correct integration of the SM; also confirmed by using a mixture of two probes hdhfr/chr.5.

Right panel: D. PbANKA-PvCSP(r)_(PbCSP): 2196cl1 and 2199cl1. the correct integration of the PvCSP expression construct into the GIMO locus was confirm by showing the removal of the hdhfr::yfcu SM cassette in the cloned chimeric parasite line (2196cl1 and 2199cl1). The southern blot is hybridized with a mixture of two probes: one recognizing hdhfr and a control probe recognizing chr-5. As an additional control (ctrl), parasite line 2117cl1 is used with the hdhfr::yfcu SM integrated into chr-3.

FIG. 4D. Genotype analysis by diagnostic PCR analysis of chimeric parasite lines confirming correct integration of both PvCSP VK210 and VK247 antigen expression cassettes. Correct integration in both lines is shown by the absence of the hdhfr::yfcu selectable marker (SM), the presence of the PvCSP gene CDS and the correct integration of the construct into the genome both at the 5′ and 3′regions (5′int and 3′int). Primers sequences used are shown in Table S1, while the expected PCR product sizes and the primer numbers are listed in the table below the Agarose gel images.

FIGS. 4E-G. Immunofluorescence assay (IFA) to assess expression of PvCSP on the surface of the transgenic sporozoites and in vivo imaging. Sporozoites from mosquito salivary-glands were stained with anti-PvCSP-210 (MR4) diluted 200 times, anti-PvCSP-247 (MR4) diluted 200 times, and anti-PbCSP (3D11) diluted 1000 times. Alexa Fluor® 488-labelled anti-mouse IgG and Hoechst-33342 were also included for the assay. (E) Transgenic parasite line 2196cl1 expressing the PvCSP VK210; (F) Transgenic parasite line 2199cl1 expressing the PvCSP VK247; (G) Wild type P. berghei parasite line 676m1cl1 showing expression of PbCSP.

FIG. 5. Fitness and phenotype analyses of two transgenic P. berghei lines expressing P. vivax CSP VK210 or VK247. Mean numbers of oocysts (A) and salivary gland sporozoites (B) in A. stephensi mosquitos at day 10 and 21, respectively (3 independent experiments; 40 mosquitoes per experiment; error bars show standard deviation). (C) Blood-stage parasitaemia (mean±SEM) in mice after intravenous (i.v.) injection of 2000 sporozoites per mouse. (D) The Kaplan-Meier curves illustrate the time to reach 1% parasitaemia (Tto1) in 6 CD1 mice after i.v. injection with 2000 transgenic or wild type sporozoites. Survival analysis was made using a Kaplan-Meier Log-rank (Mantel-Cox) test. n.s.: not significant.

FIG. 6. Viral-vectored vaccine (vv) efficacy against a challenge with transgenic P. berghei sporozoites expressing P. vivax CSP VK210 or VK247.

Groups of mice (n=6) were immunized with vv expressing PvCSP (N210/N247C, N210C, N247C, NC; see FIG. 1) using an Ad prime and MVA-boost regimen. A vv expressing P. berghei CSP was used as control. Two (A-D) or 12 (E) weeks after the boost, mice were challenged by intravenous injection of 2,000 transgenic sporozoites expressing P. vivax CSP VK210 or VK247. The Kaplan-Meier curves illustrate the time to 1% parasitaemia (A-F). As a control PbCSP or N210/N247C immunized mice were challenged with wild type P. berghei sporozoites (F). Statistical analysis was performed using the Kaplan-Meier survival curves and P values were calculated using a Kaplan-Meier Log-rank (Mantel-Cox) test. Data was obtained from two individual experiments.

FIG. 7. Development, immunogenicity and efficacy of the Rv21 virus-like particle (VLP) presenting the chimeric PvCSPVK210/247 antigen fused to the Hepatitis B Surface antigen The chimeric construct was expressed as a VLP in P. pastoris (A) Western blot to confirm expression of the VK210, VK247 and HepB surface antigen (S Ag) (B) Analysis of protein presence and purity using silver staining after affinity purification (left panels) and visualization of VLPs by transmission electron microscopy (TEM, right panels). (C) Analysis of protein purity using silver staining after a second round of purification using size exclusion chromatography and visualization of VLPs from the same purification by TEM (right panel). (D) Protective efficacy of Rv21 in CD-1 mice immunized with a low dose of 0.5 μg of VLP alone or mixed with Matrix M adjuvant and challenged with transgenic sporozoites expressing P. vivax CSP VK210 or VK247. Kaplan-Meier curves indicate the time to reach 1% parasitemia. (E) Anti-PvCSP antibody responses in the same groups of mice on week 2 after prime (day 14) or boost (day 28) and prior to challenge. (F) Protective efficacy in C57Bl/6 mice immunized with either a low dose of 0.5 μg (left) or a high dose of 5 μg (right) of Rv21 in presence or absence of Matrix M adjuvant (Adj). Mice were challenged with transgenic sporozoites expressing P. vivax CSP VK210 or VK247. The Kaplan-Meier curves illustrate the time to 1% parasitaemia. (G) Kinetic of the antibody responses in presence or absence of the Matrix M adjuvant (left) and evidence of high avidity of anti-PvCSP on the long-term with the use of Matrix M adjuvant. (H) Association (left) and correlation (right) of antibody titers with protective efficacy. (I) Association of titers of IgG2a with protection.

FIGS. 8A-8C: The effect of adjuvants and a combination of viral vectors on Rv21 anti-vivax CSP immunogenicity.

FIGS. 9A-9B: The effect of adjuvants and a combination of viral vectors on Rv21 protective efficacy against a sporozoite challenge. FIG. 9A is a graph showing the effect of the combination of Rv21+vvTRAP+Adjuvants Matrix M (MM) or Addavax (Add) against a sporozoite challenge. FIG. 9B is a graph showing the effect of a suboptimal dose of Rv21, alone or in combination with adjuvants Addavax (Add) or Matrix M (MM), against a sporozoite challenge. FIG. 9C is a graph showing the effect of a prime/boost with adenovirus-TRAP and MVA-TRAP (vvTRAP) from vivax, with or without adjuvants, against a sporozoite challenge. FIG. 9D is a graph showing the effect of vivax CSP administered as a protein (pCS) instead of a VLP composition (Rv21) with and without MM adjuvant against a sporozoite challenge.

FIG. 10: Survival data of BALB/c mice after priming with Rv21+AdTRAP and then boosting with Rv21+MVAPvTRAP.

FIG. 11: Effect of the presence of the N-terminal region of P. vivax CSP on the protective efficacy against a malaria sporozoite challenge.

FIG. 12: Characterisation of Rv21 by Western blot and ELISA.

EXAMPLES

The present invention is further illustrated by the following Examples, in which parts and percentages are by weight and degrees are Celsius, unless otherwise stated. It should be understood that these Examples, while indicating preferred embodiments of the invention, are given by way of illustration only. From the above discussion and these Examples, one skilled in the art can ascertain the essential characteristics of this invention, and without departing from the spirit and scope thereof, can make various changes and modifications of the invention to adapt it to various usages and conditions. Thus, various modifications of the invention in addition to those shown and described herein will be apparent to those skilled in the art from the foregoing description. Such modifications are also intended to fall within the scope of the appended claims.

The disclosure of each reference set forth herein is incorporated herein by reference in its entirety.

Example 1: Materials and Methods

Materials and Methods

Animals

Female inbred BALB/c (H-2^(d)), C57BL/6 (H-2^(b)) and outbred CD1 (ICR) mice were used for the assessment of immunogenicity and protection after challenge. Tuck-ordinary (TO) outbred mice were used for parasite production and transmission. Mice were purchased from Harlan (UK). Transgenic parasites were developed in Leiden University Medical Centre (LUMC) using 6-week old Swiss mice (Charles River).

Parasite Production

Wild type and transgenic parasites used to challenge mice were produced at the insectary of the Jenner Institute. Female Anopheles stephensi mosquitoes were fed on infected TO mice. Briefly, exflagellation was first confirmed and mosquitoes were exposed to anaesthetized infected mice for 10 minutes. Mosquitoes were then maintained for 21 days in a humidified incubator at a temperature of 19-21° C. on a 12 hour day-night cycle and fed with a fructose/PABA solution.

Parasites

The wild type (WT) reference line cl15cy1 of P. berghei ANKA [48] and the reporter PbANKA parasite line PbGFP-Luc_(con) (676m1cl1). PbGFP-Luc_(con) parasite expresses a fusion protein of GFP (mutant3) and firefly luciferase (LUC-IAV) under the constitutive eefla promoter and is SM free [48]. The reporter-cassette is integrated into the neutral 230p locus (PBANKA_030600). For details of PbGFP-Luc_(con), see RMgmDB entry #29 (www.pberghei.eu/index.php?rmgm=29).

Laboratory Animals and Ethics Statement

For the generation of the chimeric parasites Swiss mice (OF1 ico, Construct 242; 6 weeks old; 25-26 g; Charles River) were used. All animal experiments performed at the LUMC were approved by the Animal Experiments Committee of the Leiden University Medical Center (DEC 12042). The Dutch Experiments on Animals Act was established under European guidelines (EU directive no. 86/609/EEC regarding the Protection of Animals used for Experimental and Other Scientific Purposes).

Generation of DNA Constructs and Genotyping of the Chimeric Parasites

To generate the chimeric parasites where the P. berghei csp gene (PBANKA_040320) coding sequence (CDS) has been replaced by the CDS of P. vivax csp (PVX_119355), we used a 2-step GIMO transfection protocol [31, 49]. In the first step, we deleted the P. berghei csp CDS and replaced it with the positive-negative selectable marker, to create a P. berghei csp deletion GIMO line (PbANKA-CS GIMO). In order to do this, we generated pL1929 construct that is based on the standard GIMO DNA construct pL0034 [49]. This construct contains the positive-negative (hdhfr.:yfcu) selection marker (SM) cassette, and was used to insert both the Pbcsp 5′ and 3′ gene targeting regions (TR), encompassing the full length promoter and transcription terminators sequences respectively. The linear pL1929 DNA construct was introduced into PbGFP-Luc_(con) parasites using standard methods transfection [48]. Transfected parasites were selected in mice by applying positive selection by providing pyrimethamine in the drinking water [48]. Transfected parasites were cloned by limiting dilution [50], resulting in the PbANKA-CS GIMO line (2151cl1). Correct deletion of the P. berghei csp CDS was confirmed by diagnostic PCR-analysis on gDNA and Southern analysis of pulsed field gel (PFG) separated chromosomes as described [48]. Primers used for PCR genotyping are listed in Table S1 below.

TABLE S1 Expected PCR PCR size Primer name Primer sequence¹ product (bp) PvCSP-trans (F6) TGACATGCATATGTGTTGGTTG PvCSP 5′  744 PvCSP-trans (R5) (SEQ ID NO: 48)/ int. GCTGATTGTCCAACATGTGC (SEQ ID NO: 49) 1054 CCAAAGGAACTTAAACGAGCTATG PbCSP  744 1055 (SEQ ID NO: 50)/ CTTATACCAGAACCACATGTTACG (SEQ ID NO: 51) 7393 ATCGACTAGTAAAGCCTCGCTACG PbTRAP 1052 7394 (SEQ ID NO: 52)/ Control GTGAGTCAGATGGACTTTCTGGTAG (SEQ ID NO: 53) ¹The primer sequences (i.e. Forward/Reverse) surrounding the regulatory region are shown with the restriction site used (bold and underlined) to introduce the region into the transfection construct.

In the second step, we replaced the positive-negative SM in the PbANKA-CS GIMO genome with the CDS of either P. vivax VK210 or VK247 csp by GIMO transfection to create the two P. berghei transgenic CSP replacement lines. This was achieved by modifying the construct used in the first step (pL1929); specifically, the hdfhr::yfcu SM cassette was removed and replaced with P. vivax csp CDS sequence. The P. vivax csp CDS was ordered from GeneArt (Regensburg, Germany) (i.e. VK210) or cDNA (VK247) Both the P. vivax VK210 and VK247 CSP constructs (pL1942 and pL1943, respectively) were sequenced to ensure there were no mutations in the P. vivax csp CDS. These constructs were linearized using SacI and PacI restriction enzymes outside of the 5′ and 3′ TRs before transfection. These constructs were used to transfect parasites of the PbANKA-CS GIMO line (2151cl1) using standard methods of GIMO-transfection [30]. Transfected parasites were selected in mice by applying negative selection by providing 5-fluorocytosine (5-FC) in the drinking water of mice [51]. Negative selection results in selection of chimeric parasites where the hdhfr.:yfcu SM in the csp locus of PbANKA-CS GIMO line is replaced by the CDS of P. vivax CSP Selected chimeric parasites were cloned by the method of limiting dilution [50]. Correct integration of the constructs into the genome of chimeric parasites was analyzed by diagnostic PCR-analysis on gDNA and Southern analysis of pulsed field gel (PFG) separated chromosomes as described [48]. Primers used for PCR genotyping are listed in Table S1. This method creates chimeric ‘gene replacement’ P. berghei parasites that do not contain P. berghei csp gene CDS but express either P. vivax VK210 (PbANKA-PvCS VK210(r)_(PbCS); 2196cl1) or VK247 csp (PbANKA-PvCS VK247(r)_(PbCS); 2199cl1) under the control of the P. berghei csp regulatory sequences.

Phenotyping of Reporter and Chimeric Parasites

Growth of blood stages of the reporter and chimeric P. berghei parasites was determined during the cloning period as described [30, 48]. Feeding of A. stephensi mosquitoes, determination of oocyst production, sporozoite collection were performed as described [30]. Expression of PvCSP-VK210 and PvCSP-VK247 antigens in sporozoites of the chimeric parasites was analysed by immunofluorescence-staining assay (IFA), using anti-P. vivax antigen monoclonal antibodies (anti-PvCSP-VK210 (MR4) or anti-PvCSP-247 (MR4) antibodies; diluted 200 times) or anti-PbCSP 3D11 antibodies as a control; diluted 1000 times. Purified sporozoites were fixed with 4% paraformaldehyde in PBS for 20 min on ice, then washed three times with PBS and blocked with 20 ul 10% FCS+1% BSA in PBS for 30 min at room temperature. The excess blocking medium was removed, followed by the addition of 20-25 uL primary monoclonal antibody in 10% FCS+1% BSA in PBS (blocking medium) for 1-2 hours at room temperature or overnight at 4° C. After incubation the primary antibody was removed and the slides washed three times with PBS, followed by the staining with the secondary antibody (Alexa Fluor® 488 Goat Anti-Mouse IgG from life technologies, Cat #A-11001) diluted 800 times in 10% FCS+1% BSA in PBS (blocking medium) for 1 hour at room temperature. After washing three times with PBS, nuclei were stained with 2% Hoechst-33342 (Cell Signaling Technology #4082S) in PBS for 10 minutes at room temperature, washed twice with PBS and left to air-dry, this followed by adding Fluorescence Mounting Medium (Dako, code S3023) before complete dry out. Cover slips were mounted onto the slides, and the slides were sealed with nail polish and left to dry overnight in dark. The parasites in both blue and green channels were analyzed using a DMI-300B Leica fluorescence microscope and images processed using ImageJ software.

Antibody recognition to native, wild type P. vivax isolates from Mexico was assessed by IFA using a technique described earlier [52]. Briefly, sporozoites were produced by infection of laboratory-reared An. albimanus mosquitoes that fed on blood from patients infected with P. vivax. Mosquitoes were maintained for 15-18 days and sporozoites were collected by dissection of the salivary glands and deposited in multiwell IFAT slides at a concentration of 2,000 sporozoites/well. Slides were kept frozen at −70° C. until used. The assay was performed using sera from C57Bl/6 and CD-1 mice, which was incubated with three different batches each of sporozoites, yielding similar results between both strains. Air-dried sporozoites were also incubated with anti-VK210 or anti-VK247 monoclonal antibodies as positive controls [29]. Slides were analyzed using a confocal microscope and titers were calculated using the highest dilution that gave positive fluorescence.

Efficacy Studies: Determination of Liver Parasite Liver Load by Real Time Imaging and Determination Prepatent Period (after Challenge of Immunized Mice with Chimeric Sporozoites)

To determine the efficacy of the liver-stage vaccines, chimeric P. berghei infected A. stephensi mosquitoes were dissected 21 days post-feed and salivary gland sporozoites resuspended in RPMI-1640 media (Sigma Aldrich). 2000 sporozoites were injected i.v. into the tail vain per mouse, into both vaccinated and naïve controls.

In Vivo Imaging Using the IVIS System

All the transgenic parasite lines were generated to express the fusion protein GFP-Luciferase under the control of the constitutive eeflα promoter. The gfp-luc expression cassette is stably integrated into the Pb230p locus without introduction of a drug-selectable marker {Spaccapelo, 2010 #257; Janse, 2006 #181}. In vivo imaging of mice was performed using the IVIS 200 imaging system to determined parasite loads in livers of infected mice 44 hours post-infection {Ploemen, 2009 #258}. Mice were firstly shaved over the area of the liver, then anaesthetized and subcutaneously (s.c.) injected with 50 μl of 50 mg/ml D-luciferin substrate. Eight minutes after the injection of luciferin, mice were imaged for two minutes. Quantification of the bioluminescence signal was performed using the Living Image 4.2 image analysis software program. The readings were expressed as the total flux of photons emitted per second of exposure time.

Transgenic Parasite Fitness

Parasite fitness studies indicated similar infectivity, growth rates, gametocyte production and production of oocysts and sporozoites for the chimeric parasites expressing the P. vivax CSP genes compared to those of the WT P. berghei line. The blood stage growth and prepatent time to reach 1% parasitaemia in naïve mice for chimeric parasites were identical to the WT P. berghei line.

P. vivax CSP DNA Sequences

Four versions of ChAd63 and four of MVA were designed and produced to express various versions of the P. vivax CSP protein. One consisted of only the N- and C-terminal regions (NC); a second viral vector expressed VK210 repeats inserted in between the N- and C-terminal sequences (N210C). A third vector expressed VK247 inserted between the N- and C-terminal sequences (N247C) and a final design consisted on a chimeric VK210/247 flanked by similar N- and C-terminal repeats (sequences detailed below).

DNA transgenes were synthesized by GeneArt (Regensburg, Germany) and constructs were previously modified to improve antigen expression within the hosts cells, modifications included codon optimization for mammalian use and replacement of the endogenous PvCSP leading sequence for tPA (human plasminogen activator) (GenBank Accession no. K03021). In addition, the transmembrane GPI-anchor domain was removed from the Plasmodium vivax circumsporozoite protein (PvCSP) genes to allow protein secretion from any virus-transduced cell. The chimeric PvCSP vaccine insert consisted of the N- and C-terminal region from the Salvador I strain (NCBI Reference Sequence XP_001613068.1) flanking the central repeat regions of the circumsporozoite (CSP) VK210 of the Belem strain (GenBank accession number P08677) or VK247 of the Papua New Guinea (PNG) (GenBank accession number M69059.1). The central repeat region was designed using segments of 9 amino acid repeats as follows:

VK210 (SEQ ID NO: 3) 5x(GDRAAGQPA), (SEQ ID NO: 4) 4x(GDRADGQPA), (SEQ ID NO: 5) 1x(GNGAGGQAA)) VK247 (SEQ ID NO: 11) 2x (ANGAGNQPG/ANGAGGQAA), (SEQ ID NO: 12) 1x (ANGAGDQPG/ANGAGDQPG), (SEQ ID NO: 13) 1x (ANGADDQPG/ANGAGDQPG), (SEQ ID NO: 14) 1x (EDGAGNQPG/ANGAGDQPG)).

A region II-plus was included in the C-terminal sequence of the vaccine (EWTPCSVTCG) (SEQ ID NO: 31) [53], as well as an insertion region in the C-terminal sequence of PvCSP (GAGGQAAGGNA) (SEQ ID NO: 33) [54]. The VK210 vaccine insert consisted of the Salvador I strain sequence, while the VK247 consisted of the PNG sequence with the accession numbers mentioned above. A control viral vector lacking the expression of any transgene was used as a control in mock-vaccinated mice.

Viral Vector Construction

All of DNA constructs required for ChAd63 were cloned in two steps. In the first step, unique Acc65I and NotI sites were used to insert the synthetic transgenes into an adenovirus entry plasmid. The transgene was placed upstream of BGH poly(A) transcription termination sequence and under the control of the long cytomegalovirus (CMV) promoter (containing a regulatory element, an enhancer and an intron A). The entry plasmid also contained attachment L (attL) sequences, which were required for site-specific recombination with attachment R (attR) sites located on the destination vector.

In the second step of cloning, an in vitro Gateway reaction was performed mediated by LR Clonase II system (Invitrogen), whereby the transgene of the entry vector was integrated into the destination plasmid by site-specific recombinase through an attL-attR interaction. The diagnostic PCR was performed to confirm the desired integration before completing the production of the recombinant ChAd63.

Similarly, all of the malarial genes were inserted into an MVA shuttle vector using a similar cloning strategy. Unique Acc65I and XhoI sites were used for transgene restriction and ligation and the transgenes were inserted under the control of an endogenous P7.5 promoter. PCR and RFLP were used to verify the correct insertion before linearization of the MVA plasmid.

Rv21 HepB Surface Antigen VLP Design and Development

A gene containing a chimeric CSP sequence comprising the repeat regions VK210 and VK247, followed by the C-terminal sequence (210/247C) was designed to be fused to the Hepatitis B surface antigen (HepBsAg). The sequence was codon-optimised for optimal expression in yeast and purchased in GeneArt® (InVitrogen). DNA sequences were similar to those expressed by recombinant ChAd and MVA viruses, but without the N-terminal CSP region. The construct was cloned into the pPink-HC intracellular Pichia plasmid and amplified in E. coli. Upon plasmid linearization, four strains of Pichia pastoris (knock out for ade2, ade2-pep4, ade2-prb1, ade2-pep4-prb1) were electroporated and white colonies were selected using PAD selection plates. Protein expression was induced by addition of methanol and a kinetics analysis was used to select the highest protein production and an optimal time point. Purification of the protein/VLP was made in an affinity column in presence of imidazol, followed by gel filtration. Fractions from gel filtration and affinity column were analyzed by SDS-PAGE and subsequently transferred to nitrocellulose membranes, blocked in 5% skimmed milk/PBS followed by addition of primary and secondary antibodies. VLP-containing fractions from the gel filtration column were confirmed with anti-HepBsAg, anti-PvCSP VK210 and anti-PvCSP VK247 primary mouse antibodies, diluted in 3% BSA/PBS to 1:200, 1:20,000 and 1:20,000 respectively (MR4). A secondary donkey anti-mouse-AP conjugate in 3% BSA/PBS and Sigmafast™ BCIP/NBT tablets (Sigma-Aldrich) were used for development. Silver staining was used to analyse purity of the fractions from the FPLC column and of progressive stages of the VLP purification process. The samples were run on Mini-PROTEAN 12% gels (BioRad) with the Pierce unstained protein ladder (Thermo Scientific) run for reference. The gels were subjected to silver staining using a Pierce silver stain kit (Thermo Scientific). Direct staining of gels was carried out according to manufacturer's instructions. The particle-containing fraction from the sephacryl gel filtration column was negatively stained with 2% uranyl acetate. The sample was then imaged using a FEI Tecnai 12 Transmission Electron Microscope (TEM).

Immunization of Mice

Mice were primed with simian adenoviral vector 63 (ChAd63) encoding PvCSP at a dose of 1×10⁸ infectious units (iu) and 8 weeks later boosted with vaccinia-modified virus strain Ankara (MVA) encoding the same transgene at a concentration of 1×10⁶ plaque forming unit (pfu) per mouse. All viral vector vaccines were administered intramuscularly (i.m.) in endotoxin-free PBS. The Rv21 VLP vaccine was administered i.m. at a dose of 0.5 μg/mouse in endotoxin-free PBS.

Whole IgG Enzyme-Linked Immunosorbent Assay (ELISA)

ELISAs measuring total IgG were carried out as described previously [24]. Serum antibody endpoint titers were taken as the x-axis intercept of the dilution curve at an absorbance value three standard deviations greater than the OD405 for serum from a naïve mouse. Results were also calculated using a standardized ELISA by including a high-titre reference serum from hyper-immune mice to each ELISA plate to produce a standard curve, which in turn was used to quantify and assign ELISA units to each sample [55]. Briefly, Nunc Maxisorp Immuno ELISA plates were coated with the antigens diluted in PBS to a final concentration of 2 μg/mL. Each plate contained a standard curve, negative and positive controls, as well as serum samples of mice pre-boost (1:300) or post-boost (1:3000). An anti-mouse IgG coupled to alkaline phosphatase was used as a secondary antibody. Development was made with 4-nitrophenylphosphate diluted in diethanolamine buffer. Data was fitted to a four parameter hyperbolic curve [56].

Expression and Purification of PvCSP VK210 and VK247 Proteins

The codon optimized genes of P. vivax CSP containing VK210 or VK247 repeats were cloned into the pHLsec vector [57] with a C-terminal hexahistidine tag. Protein was expressed by transient transfection in HEK-293T cells and purified from dialyzed (against PBS buffer) conditioned medium by immobilized Co²⁺-affinity chromatography followed by size-exclusion chromatography in 20 mM Tris-HCl pH 8.0, 300 mM NaCl [40].

Peptides

Crude 20-mer peptides overlapping by 10 amino acids and representing full-lengths of P. vivax CSP VK210 and VK247 were synthesized by Mimotopes (Victoria, Australia). Individual peptide pools were used at a final concentration of 5 μg/mL.

Ex-Vivo IFN-γ EL/SPOT Assay

Ex vivo IFN-gamma (IFN-γ) ELISPOTs were carried out using PBMCs isolated from the blood as previously described [58, 59]. MAIP ELISPOT plates (Millipore) were used to plate cells. Anti-mouse IFN-γ mAb and development reagents were used according to the manufacturer specifications (Mabtech).

Intracellular Cytokine Staining (ICS)

Peripheral mononuclear cells (PBMCs) were stimulated for 5 hours in the presence of TRAP peptide pools described above. Hepatic cellular responses were assessed from perfused livers that were digested with collagenase and treated with ACK buffer to lyse red blood cells. Phenotypic and functional analysis of CD8⁺ and CD4⁺ T cells were performed using the following antibody clones: anti-CD8 PerCP-Cy5.5 (clone 53-6.7), and eFluor650 coupled anti-CD4 (GK1.5), anti-IFN-γ APC (XMG1.2), anti-TNF-α eFluor 450 (MP6-XT22), anti-IL-2 PE-Cy 7 (JES6-5H4). Flow cytometric analyses were performed using an LSRII instrument. Frequencies of cells producing cytokines in the graphs represent data where background from non-stimulated cells was subtracted. Data were analyzed with either FACSDiva or FlowJo software.

Infection of Mice

Mice were challenged with 2,000 wild type or transgenic P. berghei sporozoites. Infection was monitored from day 5 to 20 by Giemsa staining of blood smears.

Statistical Model for Parasitaemia Prediction

Percent parasitaemia was used to calculate the time required to reach a blood-stage infection of 1%, or time to 1% parasitaemia. This was predicted using a linear regression model as described previously [25]. Briefly, blood parasite counts were obtained for 3-5 consecutive days starting on day 5 after the challenge. Blood smears were stained with Giemsa, and percentages of parasitaemia calculated in all animals. The logarithm to base 10 of the calculated percentage of parasitaemia was plotted against the time after challenge and Prism 5 for Mac OS X (GraphPad software) statistical analysis package used for generating a linear regression model on the linear part of the blood-stage growth curve.

Statistical Analysis

For all statistical analyses, GraphPad Prism version 5.0 for Max OS was used unless indicated otherwise. Prior to statistical analysis to compare two or more populations, the Kolmogorov-Smirnov test for normality was used to determine whether the values followed a Gaussian distribution. An unpaired t-test was employed to compare two normally distributed groups, whereas Mann-Whitney rank test was used for comparing two non-parametric groups. If more than two groups were present non-parametric data was compared using Kruskal-Wallis test with Dunn's multiple comparison post-test, whereas normally distributed data were analyzed by one-way ANOVA with Bonferroni's multiple comparison post-test. The effect of two variables was explored using two-way ANOVA with Bonferroni's multiple comparison post-test. Correlation strength was tested using either Pearson's or Spearman's tests as indicated in the results chapters. Kaplan-Meier survival curves were used to represent protective efficacy to a challenge with any P. berghei parasite lines. All ELISA titres were also log 10 transformed before analysis. The value of p<0.05 was considered statistically significant (*p<0.05, **p<0.01, **p<0.001, and ***p<0.001).

Example 2: Cellular Immune Responses are not Induced in Mice Immunized with P. vivax Circumsporozoite Viral-Vectored Vaccines (PvCSP vv)

We first assessed whether viral-vectored vaccines (vv) expressing the P. vivax circumsporozoite protein (PvCSP) could induce cellular immune responses in inbred or outbred mouse strains. We generated four recombinant adenoviruses from chimpanzee origin (ChAd63) and four Modified Vaccinia Ankara (MVA) vv expressing various versions of PvCSP (PvCSP vv). The resulting recombinant viruses contained a series of PvCSP expression cassettes encoding the PvCSP N- and C-terminal sequences of the Salvador I strain, including or excluding certain selected central repeat sequences which are present in the two major PvCSP alleles VK210 and VK247 (FIG. 1A) [8].

First, a set of ChAd63 and MVA vv expressing only the N- and C-terminal sequences and no central repeats were generated (NC); a second set of vv contained a series of selected VK210 repeats [8] flanked by the same NC sequences (N210C); a third set expressed VK247 repeats flanked by NC (N247C). Finally we generated a ChAd63/MVA set expressing a chimeric CSP containing both type of repeats (VK210/247) flanked by NC (N210/247C). Groups of mice (n=6) received a homologous ChAd63-MVA prime-boost at an interval of eight weeks between immunizations (FIG. 1B). Cellular immune responses in mice were quantified two weeks after the final immunization using an ex vivo IFN-γ ELISpot assay and stimulating peripheral blood mononuclear cells (PBMCs) with a series of peptide pools designed to assess responses against either N210C or N247C (FIG. 1C, D). An initial experiment using the prime-boost regimen in inbred C57Bl/6 mice did not yield any positive responses (FIG. 1C, D; upper panel). This prompted the assessment of responses in additional mouse strains immunized using the same vaccination scheme. None of these additional mouse strains (C3H/He, BALB/c or outbred CD1) showed detectable antigen-specific T cell responses (FIG. 1C, D) and we concluded that the PvCSP molecules that we used in our vv do not have immunodominant T cell epitopes in the animal models tested. These results are in agreement with earlier observations made with a similar vaccine candidate expressing PvCSP for which no evidence was found for the presence of immunodominant T cell epitopes [9].

Example 3: Humoral Immune Responses are Induced in Mice Immunized with the PvCSP vv

We next assessed antibody responses induced by the ChAd63 and MVA vaccination protocols described above, using C57BL/6 mice (FIG. 1B). Serum samples were collected on fortnightly basis up until week 16 after the first immunization. We observed that immunization with the vv expressing the chimeric construct N210/247C induced antibody responses to both VK210 and the VK247 PvCSP (FIG. 2, top panel). In contrast, responses after an immunization with ChAd63 vv encoding only N210C or N247C, elicited antibodies only against their cognate version of PvCSP (FIG. 2). However, low antibody titres against the heterologous PvCSP allele were detected after a boost with the MVA vv, suggesting that several encounters with the antigen may stimulate low levels of cross-reactive antibody responses. No antibody responses were detected after a ChAd63-MVA immunization with either the NC or with vv expressing rodent P. berghei CSP (FIG. 2).

Example 4: P. vivax Sporozoites Isolated from Chiapas, Mexico are Recognized by Antibodies Induced in Mice after PvCSP vv Vaccination

We addressed the question whether mouse antibodies raised by immunisation with various PvCSP antigens encoded by the vv could recognize CSP on the surface of P. vivax sporozoites collected from patients in the endemic region of Tapachula, Chiapas [29]. We performed immunofluorescence assays (IFAs) using two types of sporozoites, VK210 and VK247 belonging to three different sporozoite batches, all yielding similar results. Results shown in FIG. 3 were obtained using sera from ChAd63-MVA PvCSP-immunized C57Bl/6 mice, but CD-1 mouse sera yielded similar results. Samples were collected on week 2 after the final boost immunization (FIG. 3). Serum from immunized mice reacted with both sporozoites expressing the VK210 allele (FIG. 3, top panel) and sporozoites expressing the VK247 allele (FIG. 3, bottom panel).

Example 5: Development of a Rodent Challenge Model for Assessing Protective Efficacy of the PvCSP Vaccination

In order to assess the in vivo protective efficacy of the P. vivax vaccines, we developed rodent challenge models, which involved creating transgenic P. berghei sporozoites where Pbcsp was replaced with either full length VK210 Pvcsp or VK247 Pvcsp. These transgenic parasites were generated using the gene insertion/marker out (GIMO) based transfection technology [30, 31]. Using this technology, we generated two Double-step Replacement (DsR) mutants [32] that resulted in the coding sequence (CDS) of P. berghei csp being replaced with the CDS of the Pvcsp in a two-step GIMO-transfection procedure (FIG. 4). First, the P. berghei csp coding sequence (CDS) was deleted by replacement with the hdhfr::yfcu selectable marker cassette (SM) (FIG. 4A, Step 1). Subsequently, in these P. berghei csp GIMO-deletion parasites the P. vivax csp CDS was inserted into the same locus thereby replacing hdhfr::yfcu with the P. vivax csp CDS (FIG. 4A, Step 2). These parasites have replaced full-length P. berghei csp CDS with that of Pvcsp and are SM free. In the two transgenic parasite lines, PbANKA-PvCS VK210(r)_(PbCS) (line 2196cl1) and PbANKA-PvCS VK247(r)_(PbCS) (line 2199cl1) the P. vivax csp gene is under the control of the P. berghei csp gene promoter (5′UTR) and transcription terminator (3′UTR) regulatory elements. In addition to expressing PvCSP, these transgenic parasites constitutively express the fusion GFP::luciferase reporter protein, which permits a determination of parasite liver-loads in the liver by real-time in vivo imaging (FIG. 4B). Correct replacement of the P. berghei csp CDS by the P. vivax csp CDS in the two transgenic lines was confirmed by diagnostic Southern analysis of chromosomes separated by pulsed-field gel electrophoresis and diagnostic PCR on genomic DNA (FIG. 4C). Immunofluorescence microscopy of sporozoites using anti-P. vivax CSP monoclonal antibodies to the repeat region of the VK210 or VK247 proteins, confirmed the expression of either PvCSP VK210 or VK247 in the two transgenic parasite lines (FIGS. 4E, F and G). The P. berghei CSP-specific monoclonal antibody, 3D11, confirmed expression P. berghei CSP in wild type P. berghei sporozoites but anti-P. berghei CSP staining was absent in either PbANKA-PvCS VK210(r)_(PbCS) or PbANKA-PvCS VK247(r)_(PbCS) sporozoites (FIG. 4) [33, 34]. We next assessed the ability of these two transgenic parasites to produce oocysts and sporozoites since replacing the endogenous csp gene with P. vivax csp could alter these characteristics. Oocyst production in A. stephensi of the two transgenic parasite lines was similar to oocyst production of WT P. berghei (FIG. 5A, p=0.24). Similarly, the PbANKA-PvCS VK210(r)_(PbCS) parasites produced salivary gland sporozoites comparable to WT parasites. In contrast, the PbANKA-PvCS VK247(r)_(PbCS) parasites showed significant (˜30%) reduction in sporozoites production compared to WT P. berghei parasites (FIG. 5B, p<0.05). The infectivity of sporozoites of both transgenic parasites was assessed by determination of the prepatent period (i.e. the time to 1% parasitaemia) after intravenous injection of 2,000 sporozoites in the tail vein of outbred CD-1 mice (Harlan, UK). All sporozoites were capable of a liver infection and, moreover, the time to 1% parasitemia was not significantly different between WT, PbANKA-PvCS VK210(r)_(PbCS) or PbANKA-PvCS VK247(r)_(PbCS) parasites (6.7, 6.5 and 6.7 days, respectively; FIG. 5C,D). These results demonstrate that transgenic P. berghei parasites expressing PvCSP alleles in place of PbCSP produce fully infectious sporozoites, which are able to complete liver stage development in mice.

Example 6: Protective Efficacy of PvCSP vv Vaccines Against a Sporozoite Challenge with P. berghei PvCSP Transgenic Parasites

We next determined vaccination efficacy by challenging vv-immunized mice with the transgenic P. berghei PvCSP transgenic parasites. These mice were immunised with the 4 different (ChAd63-MVA) PvCSP vv, as described above (FIG. 1A). Protective efficacy in these challenged mice was determined by measuring the prepatent period after intravenous injection of 2,000 P. berghei PvCSP transgenic sporozoites. Mice were monitored four days post-infection onwards by thin film blood smears stained with Giemsa. Once positive blood-films had been confirmed on three consecutive days, or mice had reached the end-point of twenty days parasite free (sterile protection), mice were sacrificed. The prepatent period, defined as the time to reach 1% parasitaemia, was subsequently calculated using a linear regression based on the three consecutive thin blood films, as described previously [25].

Our results indicated that the PvCSP central repeat sequences are necessary to generate protective immunity, as immunisations with NC only (i.e. lacking either VK210 or 247 repeat sequences) failed to induce protective immunity, demonstrated by the prepatent period being similar to mock immunized control mice (FIG. 6A). N210C immunisation resulted in partial protection against a challenge with the transgenic parasites expressing VK210, producing a significant (p=0.016) prolonged (prepatent period). In contrast, N210C immunization did not delay the time to patency of transgenic parasites expressing VK247 (FIG. 6B). Immunisation with the N247C vv did not induce protection against the transgenic parasites expressing either VK247 or VK210 (FIG. 6C). However, immunization with the chimeric N210/247C induced a significant delay in the prepatent period after challenge with both transgenic parasites expressing either VK210 (p=0.007) or VK247 (p=0.0008) and moreover, 25% and 16% of the immunized mice did not succumb to a blood stage infection with the VK210 or VK247 parasites, respectively (FIG. 6D).

While significant protective immunity was maintained 12 weeks after immunisation with the N210/247C vector as determined by a prolonged prepatent period after challenge with either VK210 (p=0.005) or VK247 (p=0.016) transgenic sporozoites, sterile protection was not induced as all mice eventually developed a blood stage infection (FIG. 6E). We confirmed the absence of unspecific protection induced by immunisation with the N210/247C vv vaccination through challenge of the immunised mice with wild-type P. berghei sporozoites. These mice were not protected and protection against wild-type P. berghei sporozoites was only induced in mice after a prime-boost (ChAd63-MVA) immunisation regime with a vv expressing P. berghei CS protein (p=0.024; FIG. 6F). These results demonstrate that immune responses to the central PvCSP repeats are necessary to induce protective immune responses against infection. Moreover, a chimeric VK210/247 PvCSP vv containing repeats from both forms of PvCSP elicits protective immune responses against sporozoites expressing either VK210 or VK247, which are at least equal to if not better than after immunisation with a vaccine consisting of only one form of PvCSP.

Example 7: Vaccination with the Rv21 VLP, Presenting the Chimeric PvCSP VK210/247 Antigen Fused to the Hepatitis B Surface (HepB S), Antigen Induces High Levels of Protective Immunity in Mice

The ability to induce antibody-mediated protective immunity by the PvCSP vv in the absence of detectable cellular responses led us to develop a VLP vaccine platform with a greater potential to enhance humoral responses against PvCSP. VLPs are known for their ability to induce high antibody titres, and are a leading vaccine platform not only for malaria [5] but also for HPV [35]. RTS,S/AS is based on the hepatitis B surface antigen virus-like particle (VLP) platform, genetically-engineered to include the central repeats and carboxy (C-) terminus (amino acids 207-395) of the P. falciparum CSP antigen [36]. We developed a VLP consisting of the chimeric PvCSP VK210/VK247 central repeats and the CSP C-terminal sequence fused to the Hepatitis B Surface Antigen (HepB-S) gene. Codon usage of the fusion gene was optimized for expression in Pichia pastoris and production of the fusion protein (PvCSP-HepB-S) was assessed in four yeast strains using a kinetics analysis.

We selected a P. pastoris colony that had optimal expression levels, which peaked after 108 hours (FIG. 7A). Presence of the fusion protein PvCSP-HepB S was confirmed by Western blot analyses using antibodies against PvCSP VK210, PvCs VK247 and HepB S (FIG. 7B). Presence, size and purity of the Rv21 protein was carried out using a sensitive silver stain technique (FIG. 7C, D left panel).

The protocol for purification of the fusion protein involved two steps. The first consisted of an affinity purification using a capture select C-tag molecule (Thermofisher) bound to the fusion protein. In addition to collecting the expected protein band corresponding to the PvCSP-HepB S protein, the sample also contained additional proteins. VLP particle formation was confirmed using transmission electron microscopy (TEM; FIG. 7C, right panel). A subsequent purification step was performed by size exclusion chromatography in order to obtain a single protein band of expected size of the PvCSP-HepB S protein (75 kDa), which was visualized using the silver staining technique. A further TEM analysis confirmed a cleaner preparation of VLPs (FIG. 7D, right panel). In FIG. 7E, a diagram is shown with the proposed structure of the VLP, which we have named Rv21. The purified Rv21 was used to immunize mice, using a low dose of 0.5 pg and employing a homologous prime-boost immunization protocol (FIG. 7F). Instead of the AS01 adjuvant, standardly used for RTS,S vaccination, Matrix-M adjuvant was used to enhance the immunogenicity and protective efficacy of Rv21. Matrix-M is suitable for human use and consists of saponin-based 40 nm particles that can activate and recruit immune cells to the draining lymph nodes and spleen [37, 38]. This adjuvant functions by inducing high titres of reactive antibodies supported by a balanced Th1/Th2 response [39].

The protective efficacy of each prime-boost regimen was assessed by challenging the Rv21-immunized CD-1 mice with a dose of 2000 PbANKA-PvCS VK210(r)_(PbCS) or PbANKA-PvCS VK247(r)_(PbCS) transgenic sporozoites (FIG. 7G). Protective efficacy in these immunized mice was determined by measuring the prepatent period as described above. At the low dose (0.5 μg/mouse) of the Matrix-M-adjuvanted Rv21 prime-boost immunization, protective efficacy against challenge with PbANKA-PvCS VK210(r)_(PbCS) sporozoites was 100% and against PbANKA-PvCS VK247(r)_(PbCS) sporozoites 90%, as confirmed by the absence of a blood infection 20 days after challenge (FIG. 7G). In contrast, all mice in the naïve group developed blood stage parasitemia with a prepatent period of 4-5 days. In the absence of the Matrix-M adjuvant, immunisation of mice using the VLP homologous prime-boost regime still generated sterile immunity in 50% of the VLP-immunized mice challenged with PbANKA-PvCS VK210(r)_(PbCS) sporozoites and 43% of immunized mice were protected against a challenge of PbANKA-PvCS VK247(r)_(PbCS) sporozoites (FIG. 7G).

These results indicated the importance of both the VLP and the adjuvant in inducing protective immune responses of this vaccination regime. In addition, our results showed that PvCSP VLP-based vaccination induced higher protective efficacy than immunisation with the viral-vectored vaccine candidates. ELISA analyses of antibody responses to vaccination supported the importance of Matrix-M adjuvant to elicit high antibody titres to PvCSP, even after a single (priming) immunisation with Rv21 (FIG. 7H). A prime-boost regimen with Rv21 in the absence of the Matrix-M adjuvant induced higher titres than those by a single vaccination but still lower compared to the prime-boost regime with the adjuvant. We concluded that the Rv21 antibody responses benefit from the co-administration of an adjuvant, both in a single immunization or a prime-boost regime.

We tested Rv21 efficacy in C57BL/6, a mouse strain that has previously shown to be highly sensitive to a P. berghei sporozoite infection and for which we have previously failed to induce sterile immunity using a P. vivax TRAP vaccine candidate [40]. Immunisation of mice with an Rv21 dose as described above (0.5 μg) followed by a challenge with 2,000 transgenic PbANKA-PvCS VK210(r)_(PbCS) sporozoites failed to show protective efficacy (FIG. 7I). However, a 10× increase in the Rv21 dose (5 μg) showed, for the first time in our hands, complete protection in C57BL/6 mice even against a challenge with a higher dose of 5,000 PbANKA-PvCS VK210(r)_(PbCS) sporozoites (FIG. 7I). Rv21 immunization (in Matrix M) induced antibody titres that remained consistently high for up to 70 days and showed a good positive correlation with protection (r=0.54; p=0.0018), while addition of the chaotropic agent urea for the ELISA, indicated that the inclusion of Matrix M adjuvant enhanced antibody avidity, as the addition of urea decreased antibody titres by 26.2% when using adjuvant and 100% in its absence (FIG. 7J). The IgG2a isotype levels were associated with protection (FIG. 7K), while other isotypes (IgM, IgG1,IgG3) were not (data not shown).

Our results indicate that immunization with a chimeric VK210/247 vaccine can induce immunity to the two major strains of P. vivax, based on the type of PvCSP central repeats. Moreover, high levels of protective immunity levels can be achieved using a VLP platform in presence of the Matrix-M adjuvant, even in a mouse strain that is difficult to protect against a sporozoite infection.

Example 8: The Effect of Adjuvants and a Combination of Viral Vectors on Rv21 Anti-Vivax CSP Immunogenicity

BALB/c mice were immunized using two consecutive doses (prime-boost) of Rv21-VLP presenting vivax CSP on the surface. The results are shown in FIG. 8. In FIG. 8A, one group received only Rv21 (thin dotted line), while another group received a combination of Rv21+viral vectors expressing TRAP (vvTRAP), consisting of a prime with Rv21+Adenovirus expressing vivax TRAP (vvTRAP) and a boost eight weeks later with MVA expressing vivax TRAP mixed with Rv21 (black line with squares). Anti-CSP antibodies were significantly higher with the Rv21+vvTRAP immunisation regime. In FIG. 8B, Rv21 was administered twice either alone (Rv21), or mixed with Addavax adjuvant or using a triple combination with Rv21+Addavax+vvTRAP viral vectors. Responses were significantly higher with Addavax or Rv21+Add+vvTRAP compared to Rv21 alone. In FIG. 8C, Rv21 was administered twice either alone (Rv21), or mixed with Matrix M (MM) adjuvant or using a triple combination with Rv21+MM+vvTRAP viral vectors. Responses were significantly higher with MM or Rv21+MM+vvTRAP compared to Rv21 alone.

Example 9: The Effect of Adjuvants and a Combination of Viral Vectors on Rv21 Protective Efficacy Against a Sporozoite Challenge

BALB/c mice were immunized using two consecutive doses (prime-boost) of Rv21-VLP presenting vivax CSP on the surface, or a combination with Rv21+Adjuvant+vvTRAP (viral vectors expressing vivax TRAP). The results are shown in FIG. 9. In FIG. 9A, the combination of Rv21+vvTRAP+Adjuvants Matrix M (MM) or Addavax (Add) provided 100% of sterile, complete protection against a sporozoite challenge, evidenced as the complete absence of blood stage parasitaemia during 20 days, when naïve mice succumbed to disease on days 5-7. In FIG. 9B, at the suboptimal dose tested, Rv21 afforded 50% protection while the inclusion of adjuvants Addavax (Add) or Matrix M (MM) protected 83.3% and 100% of mice against a sporozoite challenge. In FIG. 9C, a prime/boost with adenovirus-TRAP and MVA-TRAP (vvTRAP) from vivax does not confer protection at all, with or without adjuvants. In FIG. 9D, when the vivax CSP is administered as a protein (pCS) instead of a VLP composition (Rv21), only 33% of mice are protected but only upon addition of MM adjuvant, compared to 100% of Rv21.

Example 10: A Combination of Rv21+Viral Vectors (vv) Require Both Antigens to be from Vivax to Induce Complete Protection Against a Sporozoite Challenge

The results of this example are shown in FIG. 10 and in the Table below. In the Table below, mice were vaccinated with a combination of Rv21+viral vectors (vv) in a prime/boost regimen consisting of Rv21+AdenovirusVivaxTRAP for priming and Rv21+MVAVivaxTRAP for boosting (Rv21/vvTRAP); Rv21+AdControl for priming and Rv21+MVA control for boosting (Rv21/vvControl), where controls express an unrelated T. cruzi antigen); R21+AdVivaxTRAP (falciparum) for priming and R21+MVAVivaxTRAP (falciparum) for boosting (R21Control/vvTRAP).

Ad MVA Ad MVA Rv21 TRAP TRAP R21 Tc24 Tc24 Group μg iu pfu μg iu pfu Rv21/ 1 μg — — — 1 × 10⁸ 1 × 10⁷ vvControl R21control/ — 1 × 10⁸ 1 × 10⁷ 1 μg vvTRAP Rv21/ 1 μg 1 × 10⁸ 1 × 10⁷ — — — vvTRAP naïve — — — — — —

In FIG. 10, complete, 100% sterile protection against a malaria challenge was achieved with Rv21/vvTRAP, indicating that both antigens need to be from P. vivax to induce complete protection. Rv21+vvControl induced only 50% protection while vvTRAP+R21control did not induce any sterile protection, only a delay in infection.

Example 11: Effect of the Presence of the N-Terminus Region of P. vivax CSP on the Protective Efficacy Against a Malaria Sporozoite Challenge

Two forms of P. vivax CSP were produced: a full version containing the N-terminus, repeats of VK210 and VK247 and C-terminus region (FIG. 11A); and a truncated version without the N-terminus region (FIG. 11B). Proteins were used to immunize mice and a challenge with a transgenic parasite expressing the vivax CSP was performed. Protective efficacy was complete when using a truncated form of the protein, whereas the presence of the N-terminus provoked a significant decrease in protection, indicating that the lack of the N-terminus can benefit protective efficacy.

Example 12: Additional Characterization of Rv21 by Western Blot and ELISA

The Rv21 VLP particle (FIG. 12A) was produced in P. pastoris yeast, purified by affinity chromatograpy and a series of western blots were performed for further characterization and confirmation of ability to bind antibodies against P. vivax CSP. FIG. 12B shows a PAGE Silver stain under reducing conditions using decreasing amounts of Rv21. FIG. 12C shows a contrast between reducing and non-reducing conditions and binding to a monoclonal antibody against vivax CSP VK210 repeats—one of the major circulating strains around the world. FIG. 12D shows recognition of Rv21 by various samples including immune sera from mice previously vaccinated with Rv21 (left), monoclonal anti-PvCSPVK210 (middle left), monoclonal anti-PvCSPVK247 (middle right), and immune mouse sera previously vaccinated with HepB surface Antigen, which is the platform used for Rv21. VK247 corresponds to one of the two major vivax strains circulating around the world, and thus results indicate Rv21 could provide coverage against those two major strains. FIG. 12E shows recognition of Rv21 by monoclonal anti-CSPVK210, anti-CSPVK247 and mouse sera. The latter seems to recognize Rv21 at even lower dilutions, probably due to the presence of both, anti-VK210 and anti-VK247 antibodies.

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Reyes-Sandoval, A., et al., Single-dose immunogenicity and     protective efficacy of simian adenoviral vectors against Plasmodium     berghei. Eur J Immunol, 2008. 38(3): p. 732-41. -   60. Shoukat et al., Journal of Infectious Disease 1993, 168:1485-9     Additional Sequences

Type I VK210 wild type sequence. P. vivax CSP Belem strain P08677 (ref. sequence from www.uniprot.org) SEQ ID NO: 1 MKNFILLAVSSILLVDLFPTHCGHNVDLSKAINLNGVNFNNVDASSLGAAHVGQSASRGRGLGE NPDDEEGDAKKKKDGKKAEPKNPRENKLKQPGDRADGQPAGDRADGQPAGDRADGQPAGDRAAG QPAGDRADGQPAGDRADGQPAGDRADGQPAGDRADGQPAGDRAAGQPAGDRAAGQPAGDRADGQ PAGDRAAGQPAGDRADGQPAGDRAAGQPAGDRADGQPAGDRAAGQPAGDRAAGQPAGDRAAGQP AGDRAAGQPAGNGAGGQAAGGNAGGGQGQNNEGANAPNEKSVKEYLDKVRATVGTEWTPCSVTC GVGVRVRRRVNAANKKPEDLTLNDLETDVCTMDKCAGIFNVVSNSLGLVILLVLALFN. Type II VK247 wild type sequence. P. vivax CSP PND (Papua New Guinea) strain Q7M3X0 (ref. sequence from www.uniprot.org) SEQ ID NO: 2 MKNFILLAVSSILLVDLFPTHCGHNVDLSKAINLNGVGFNNVDASSLGAAHVGQSASRGRGLGE NPDDEEGDAKKKKDGKKAEPKNPRENKLKQPEDGAGNQPGANGAGNQPGANGAGNQPGANGAGD QPGANGAGNQPGANGAGDQPGANGAGNQPGANGAGNQPGANGAGNQPGANGADDQPGANGAGNQ PGANGAGNQPGANGAGNQPGANGAGDQPGANGAGNQPGANGAGDQPGANGAGNQPGANGAGNQP GANGAGNQPGANGAGNQPGANGAGGQAAGGNAANKKAGDAGAGQGQNNEGANATNEKSVKEYLD KVRATVGTEWTPCSVTCGVGVRVRRRVNAANKKPEDLTLNDLETDVCTMDKCAGIFNVVSNSLG LVILLVLALFN Pv VK210 Repeats DNA Sequence (SEQ ID NO: 15) GGTGATAGAGCTGCTGGTCAACCTGCTGGTGACAGAGCTGCTGGACAGCCAGCTGGTGATAGAG CTGCTGGTCAGCCTGCTGGTGATAGAGCTGCTGGACAACCTGCTGGTGATAGAGCTGCTGGTCA ACCTGCTGGTGATAGAGCTGACGGTCAGCCAGCTGGTGATAGAGCTGACGGTCAACCTGCTGGT GACAGAGCTGACGGACAACCTGCTGGTGATAGAGCTGATGGACAACCAGCTGGAAATGGTGCTG GTGGTCAAGCTGCT Amino acid sequence (SEQ ID NO: 16) GDRAAGQPAGDRAAGQPAGDRAAGQPAGDRAAGQPAGDRAAGQPAGDRADGQPAGDRADGQPAG DRADGQPAGDRADGQPAGNGAGGQAA Pv VK247 Repeats DNA Sequence (SEQ ID NO: 17) GCTAATGGTGCTGGAAATCAACCAGGTGCTAACGGTGCTGGTGGACAGGCTGCTGCTAACGGTG CTGGTAACCAGCCTGGTGCTAATGGTGCTGGTGGACAAGCTGCTGCTAACGGTGCTGGTGATCA ACCAGGTGCTAATGGTGCTGGTGATCAGCCTGGTGCTAACGGTGCTGATGACCAACCTGGTGCT AACGGTGCTGGTGACCAGCCAGGTGAGGACGGTGCTGGTAATCAACCTGGTGCTAACGGTGCTG GTGATCAACCTGGT Amino acid sequence (SEQ ID NO: 18) ANGAGNQPGANGAGGQAAANGAGNQPGANGAGGQAAANGAGDQPGANGAGDQPGANGADDQPGA NGAGDQPGEDGAGNQPGANGAGDQPG P vivax-like repeats Protein ID: UniProtKB-Q26124 (Q26124_9APIC) Repeat I DNA Sequence (SEQ ID NO: 22) gccccaggagcaaatcaggaaggtggagcagcagccccaggagcaaatcaggaaggtggagcag cagccccaggagcaaatcaggaaggtggagcagca Amino acid sequence (SEQ ID NO: 23) APGANQEGGAAAPGANQEGGAAAPGANQEGGAA P. vivax-like repeat II DNA Sequence (SEQ ID NO: 24) gcaccaggagcaaaccagggaggtggagcagcagcaccaggagcaaaccagggaggtggagcag cagcaccaggagcaaaccagggaggtggagcagca Amino acid sequence (SEQ ID NO: 25) APGANQGGGAAAPGANQGGGAAAPGANQGGGAA P. vivax-like repeat I + P. vivax-like repeat II Amino acid sequence (SEQ ID NO: 26) APGANQEGGAAAPGANQEGGAAAPGANQEGGAA APGANQGGGAAAPGANQGGGAAAPGANQGGGAA P. vivax-like repeat II + P. vivax-like repeat I Amino acid sequence (SEQ ID NO: 27) APGANQGGGAAAPGANQGGGAAAPGANQGGGAA APGANQEGGAAAPGANQEGGAAAPGANQEGGAA C-Terminus of PvCSP. Protein ID in UNIPROT: UniProtKB-P08677 (CSP_PLAVB) DNA Sequence (SEQ ID NO: 28) CAAGCTGCTGGTGGTAATGCTGGTGGTGGTCAGGGTCAAAACAACGAGGGTGCTAATGCTCCAA ACGAGAAGTCCGTTAAGGAATACTTGGACAAAGTTAGAGCTACTGTTGGTACTGAGTGGACTCC ATGTTCCGTTACTTGTGGTGTTGGTGTTAGAGTTAGAAGAAGAGTTAACGCTGCTAACAAGAAG CCAGAGGACTTGACTTTGAACGACTTGGAGACTGACGTTTGTACTATGGACAAG Protein Sequence (SEQ ID NO: 29) QAAGGNAGGGQGQNNEGANAPNEKSVKEYLDKVRATVGTEWTPCSVTCGVGVRVRRRVNAANKK PEDLTLNDLETDVCTMDK C-Terminus fragment of PvCSP Amino acid sequence (SEQ ID NO: 30) EWTPCSVTCGVGVRVRRRVNAANKKPEDLTLNDLETDVCTMDK N-terminus sequence from PvCSP in Adenovirus. Gene ID PVX_187290. Plasmodium vivax (strain Salvador I), UNIPROT UniProtKB-A5KDP2 (A5KDP2_PLAVS) DNA Sequence (SEQ ID NO: 34) ACCCACTGCGGCCACAACGTGGACCTGAGCAAGGCCATCAACCTGAACGGCGTGAACTTCAACA ATGTGGACGCCTCTAGCCTGGGAGCTGCTCACGTGGGCCAGAGCGCCAGCAGAGGCAGAGGCCT GGGCGAGAACCCCGACGATGAGGAAGGCGACGCCAAGAAGAAGAAGGACGGCAAGAAGGCCGAG CCCAAGAACCCCAGAGAGAACAAGCTGAAGCAGCCC Amino acid sequence (SEQ ID NO: 35) THCGHNVDLSKAINLNGVNFNNVDASSLGAAHVGQSASRGRGLGENPDDEEGDAKKKKDGKKAE PKNPRENKLKQP Hepatitis B S Antigen DNA sequence (SEQ ID NO: 37) CCAGTTACTAATATGGAAAACATCACTTCCGGTTTCTTGGGTCCTTTGTTGGTTTTGCAGGCTG GATTCTTCTTGTTGACTAGAATCTTGACTATCCCACAGTCCTTGGACTCTTGGTGGACTTCCTT GAACTTCTTGGGTGGTTCCCCAGTTTGTTTGGGTCAGAACTCTCAATCCCCAACTTCTAACCAC TCCCCAACATCCTGTCCTCCAATTTGTCCAGGTTACAGATGGATGTGTTTGAGAAGATTCATCA TTTTCTTGTTCATCTTGTTGTTGTGTTTGATCTTCTTGTTGGTTTTGTTGGACTACCAGGGTAT GTTGCCAGTTTGTCCATTGATCCCAGGTTCCACTACTACAAACACTGGTCCATGTAAGACTTGT ACTACTCCAGCTCAGGGTAACTCTATGTTCCCTTCCTGTTGTTGTACTAAGCCAACTGACGGTA ACTGTACTTGTATCCCAATTCCATCCTCCTGGGCTTTCGCTAAGTACTTGTGGGAATGGGCTTC CGTTAGATTCTCTTGGTTGTCCTTGTTGGTTCCATTCGTTCAGTGGTTCGTTGGTTTGTCCCCA ACTGTTTGGTTGTCTGCTATCTGGATGATGTGGTACTGGGGTCCATCCTTGTACTCTATCGTTT CCCCATTCATCCCTTTGTTGCCAATCTTCTTCTGTTTGTGGGTTTACATC Amino acid sequence (SEQ ID NO: 38) PVTNMENITSGFLGPLLVLQAGFFLLTRILTIPQSLDSWWTSLNFLGGSPVCLGQNSQSPTSNH SPTSCPPICPGYRWMCLRRFIIFLFILLLCLIFLLVLLDYQGMLPVCPLIPGSTTTNTGPCKTC TTPAQGNSMFPSCCCTKPTDGNCTCIPIPSSWAFAKYLWEWASVRFSWLSLLVPFVQWFVGLSP TVWLSAIWMMWYWGPSLYSIVSPFIPLLPIFFCLWVYI Preferred Rv21 sequence Amino acid sequence SEQ ID NO: 39 (SEQ ID NOs: 16 + 18 + 29 + 38) GDRAAGQPAGDRAAGQPAGDRAAGQPAGDRAAGQPAGDRAAGQPAGDRADGQPAGDRADGQPAG DRADGQPAGDRADGQPAGNGAGGQAA ANGAGNQPGANGAGGQAAANGAGNQPGANGAGGQAAANGAGDQPGANGAGDQPGANGADDQPGA NGAGDQPGEDGAGNQPGANGAGDQPG QAAGGNAGGGQGQNNEGANAPNEKSVKEYLDKVRATVGTEWTPCSVTCGVGVRVRRRVNAANKK PEDLTLNDLETDVCTMDK PVTNMENITSGFLGPLLVLQAGFFLLTRILTIPQSLDSWWTSLNFLGGSPVCLGQNSQSPTSNH SPTSCPPICPGYRWMCLRRFIIFLFILLLCLIFLLVLLDYQGMLPVCPLIPGSTTTNTGPCKTC TTPAQGNSMFPSCCCTKPTDGNCTCIPIPSSWAFAKYLWEWASVRFSWLSLLVPFVQWFVGLSP TVWLSAIWMMWYWGPSLYSIVSPFIPLLPIFFCLWVYI PvTRAP sequence from SalI strain of P. vivax (wild-type) Protein sequence (SEQ ID NO: 40) MKLLQNKSYLLVVFLLYVSIFARGDEKVVDEVKYSEEVCNESVD LYLLVDGSGSIGYPNWITKVIPMLNGLINSLSLSRDTINLYMNLFGNYTTELIRLGSG QSIDKRQALSKVTELRKTYTPYGTTNMTAALDEVQKHLNDRVNREKAIQLVILMTDGV PNSKYRALEVANKLKQRNVSLAVIGIGQGINHQFNRLIAGCRPREPNCKFYSYADWNE AVALIKPFIAKVCTEVERVANCGPWDPWTACSVTCGRGTHSRSRPSLHEKCTTHMVSE CEEGECPVEPEPLPVPAPLPTVPEDVNPRDTDDENENPNENKGLDVPDEDDDEVPPAN ERADGNPVEENVFPPADDSVPDESNVLPLPPAVPGGSSEEFPADVQNNPDSPEELPME QEVPQDNNVNEPERSDSKGYGVNEKVIPNPLDNERDMANKNKTVHPGRKDSARDRYAR PHGSTHVNNNRANENSDIPNNPVPSDYEQPEDKAKKSSNNGYKIAGGVIAGLALVGCV GFAYNFVAGGGAAGMAGEPAPFDEAMAEDEKDVAEADQFKLPEDNEWN PvTRAP variant sequence Protein sequence (SEQ ID NO: 41) DEKVVDEVKYSEEVCNESVDLYLLVDGSGSIGYPNWITKVIPMLNGLINSLSLSRDTINLYMNL FGNYTTELIRLGSGQSIDKRQALSKVTELRKTYTPYGTTNMTAALDEVQKHLNDRVNREKAIQL VILMTDGVPNSKYRALEVANKLKQRNVSLAVIGVGQGINHQFNRLIAGCRPREPNCKFYSYADW NEAVALIKPFIAKVCTEVERVANCGPWDPWTACSVTCGRGTHSRSRPSLHEKCTTHMVSECEEG ECPVEPEPLPVPAPLPTVPEDVNPRDTDDENENPNFNKGLDVPDEDDDEVPPANEGADGNPVEE NVFPPADDSVPDESNVLPLPPAVPGGSSEEFPADVQNNPDSPEELPMEQEVPQDNNVNEPERSD SNGYGVNEKVIPNPLDNERDMANKNKTVHPGRKDSARDRYARPHGSTHVNNNRANENSDIPNNP VPSDYEQPEDKAKKSSNNGYK C-tag DNA sequence (SEQ ID NO: 42) GAGCCCGAGGCC Amino acid sequence (SEQ ID NO: 43) EPEA Pvs25 DNA sequence (SEQ ID NO: 44) ATGgccgtcacggtagacaccatatgcaaaaatggacagctggttcaaatgagtaaccacttta agtgtatgtgtaacgaagggctggtgcacctttccgaaaatacatgtgaagaaaaaaatgaatg caagaaagaaaccctaggcaaagcatgcggggaatttggccagtgtatagaaaacccagaccca gcacaggtaaacatgtacaaatgtggttgcattgagggctacactttgaaggaagacacttgtg tgcttgatgtatgtcaatacaaaaattgtggagaaagtggcgaatgcattgttgagtacctctc ggaaatccaaagtgcaggttgctcatgtgctattggcaaagtccccaatccagaagatgagaaa aaatgtaccaaaacgggagaaactgcttgtcaattgaaatgtaacacagataatgaagtctgca aaaatgttgaaggagtttacaagtgccagtgtatggaaggctttacgttcgacaaagagaaaaa tgtatgcctttcctattctgtatttaacatcctaaactactccctcttctttatcatcctgctt gtcctttcgtacgtcatataa Pvs25 Amino acid sequence (SEQ ID NO: 45) MAVTVDTICKNGQLVQMSNHFKCMCNEGLVHLSENTCEEKNECKKETLGKACGEFGQCIENPDP AQVNMYKCGCIEGYTLKEDTCVLDVCQYKNCGESGECIVEYLSEIQSAGCSCAIGKVPNPEDEK KCTKTGETACQLKCNTDNEVCKNVEGVYKCQCMEGFTFDKEKNVCLSYSVFNILNYSLFFIILL VLSYVI Pvs28 Amino acid sequence (SEQ ID NO: 46)                        MKVTAETQC KNGYVVQMSN HFECKCNDGF                               80         90        100 VMANENTCEE KRDCTNPQNV NKNCGDYAVC ANTRMNDEER ALRCGCILGY        110        120        130        140        150 TVMNEVCTPN KCNGVLCGKG KCILDPANVN STMCSCNIGT TLDESKKCGK        160        170        180        190        200 PGKTECTLKC KANEECKETQ NYYKCVAKGS GGEGSGGEGS GGEGSGGEGS        210        220        230 GGEGSGGDTG AAYSLMNGSA VISILLVFAF FMMSLV Pvs25 + Pvs28 fusion Amino acid sequence (SEQ ID NO: 47) MAVTVDTICKNGQLVQMSNHFKCMCNEGLVHLSENTCEEKNECKKETLGKACGEFGQCIENPDP AQVNMYKCGCIEGYTLKEDTCVLDVCQYKNCGESGECIVEYLSEIQSAGCSCAIGKVPNPEDEK KCTKTGETACQLKCNTDNEVCKNVEGVYKCQCMEGFTFDKEKNVCLSYSVFNILNYSLFFIILL VLSYVIKVTAETQCKNGYVVQMSNHFECKCNDGFVMANENTCEEKRDCTNPQNVNKNCGDYAVC ANTRMNDEERALRCGCILGYTVMNEVCTPNKCNGVLCGKGKCILDPANVNSTMCSCNIGTTLDE SKKCGKPGKTECTLKCKANEECKETQNYYKCVAKGSGGEGSGGEGSGGEGSGGEGSGGEGSGGD TGAAYSLMNGSAVISILLVFAFFMMSLV 

The invention claimed is:
 1. A fusion polypeptide or a particle comprising the fusion polypeptide, wherein the fusion polypeptide comprises: (i) the Plasmodium vivax Type I circumsporozoite protein (CSP) repeat units: (SEQ ID NO: 3) (GDRAAGQPA)*5 (SEQ ID NO: 4) (GDRADGQPA)*4 (SEQ ID NO: 5) (GNGAGGQAA)*1 (SEQ ID NO: 11) (ANGAGNQPG-ANGAGGQAA)*2 (SEQ ID NO: 12) (ANGAGDQPG-ANGAGDQPG)*1 (SEQ ID NO: 13) (ANGADDQPG-ANGAGDQPG)*1 and (SEQ ID NO: 14) (EDGAGNQPG-ANGAGDQPG)*1

(ii) the Plasmodium vivax Type II circumsporozoite (CSP) repeat units: (ANGAGNQPG-ANGAGGQAA)*2 (SEQ ID NO: 11) (ANGAGDQPG-ANGAGDQPG)*1 (SEQ ID NO: 12) (ANGADDQPG-ANGAGDQPG)*1 (SEQ ID NO: 13) and (EDGAGNQPG-ANGAGDQPG)*1 (SEQ ID NO: 14)

wherein the above Type I and Type II repeat units are joined contiguously in the above order in a N-C orientation, and *5 represents multiplied by 5, *4 represents multiplied by 4, *2 represents multiplied by 2, and *1 represents multiplied by 1; and (iii) (a) an amino acid sequence having at least 80% sequence identity to one or more of SEQ ID NOs: 29-32; or (b) a fragment of SEQ ID NOs: 29-32 which is at least 50% of the length of SEQ ID NOs: 29-32.
 2. The fusion polypeptide or particle as claimed in claim 1, wherein the fusion polypeptide additionally comprises: (iv) at least one repeat unit of a circumsporozoite protein (CSP) of a Plasmodium vivax-like malaria parasite, wherein the amino acid sequence of the at least one repeat unit is APGANQ(E/G)GGAA (SEQ ID NO: 19).
 3. The fusion polypeptide or particle as claimed in claim 1, wherein the fusion polypeptide additionally comprises: (v) (a) an amino acid sequence having at least 80% sequence identity to SEQ ID NO: 38; or (b) a fragment of SEQ ID NO: 38 which is at least 80% of the length of SEQ ID NO:
 38. 4. A pharmaceutical composition comprising the fusion polypeptide or particle as claimed in claim 1, optionally together with one or more pharmaceutically-acceptable carriers, excipients or diluents.
 5. A vaccine composition comprising the fusion polypeptide or particle as claimed in claim 1, together with a pharmaceutically-acceptable adjuvant. 